@article{tsuji_ninomiya-tsuji_shen_difrancesco_2025, title={Modulation of iron metabolism by new chemicals interacting with the iron regulatory system}, volume={79}, ISSN={["2213-2317"]}, DOI={10.1016/j.redox.2024.103444}, journal={REDOX BIOLOGY}, author={Tsuji, Yoshiaki and Ninomiya-Tsuji, Jun and Shen, Maurice Y. F. and Difrancesco, Benjamin R.}, year={2025}, month={Feb} }
 @article{gonzalez-calderon_lopez-perez_sai_ninomiya-tsuji_2024, title={TAK1 inhibition translocates pore-forming proteins, MLKL and gasdermins into mitochondria to generate reactive oxygen species}, volume={300}, ISSN={["1083-351X"]}, DOI={10.1016/j.jbc.2024.106731}, abstractNote={Mitogen-activated protein kinase kinase kinase 7 (MAP3K7), known as TAK1, is a central mediator of intracellular host defense signaling promoting inflammatory gene expression. Hence, TAK1 is a prime target of intracellular bacterial effectors in blocking inflammatory gene expression. However, when TAK1 is inhibited, host cells alternatively induce mitochondrial reactive oxygen species (ROS) to kill intracellular bacteria, which ultimately induces host cell death to prevent bacteria spreading. In the current study, we delineate the mechanism of how host cells elevate ROS and induce cell death in response to TAK1 inhibition in bone marrow derived macrophage (BMDMs). TAK1 inhibition activates multiple cell death pathways, namely caspase 8-dependent apoptosis and pyroptosis, and receptor interacting protein kinase 3 (RIPK3)-dependent necroptosis. While these pathways lead to host cell death, we found that mixed lineage kinase-like (MLKL) downstream RIPK3 and gasdermins (GSDMD and GSDME) downstream caspase 8 were translocated to mitochondria upon TAK1 inhibition. Deletion of both MLKL and gasdermins partially blocked TAK1 inhibition-induced mitochondrial ROS and exacerbated intracellular bacteria growth colonization. These results suggest that these cell-killing pore forming proteins play an alternative host defense role to prevent intracellular pathogen colonization by modulating mitochondria. Additionally, we found that ablation of both caspase 8 (apoptosis and pyroptosis) and RIPK3 (necroptosis) did not abolish TAK1 inhibition-induced cell death in BMDMs. Furthermore, we found that mice having TAK1, caspase 8 and RIPK3 triple deletion died within 8 days. These results suggest that an unidentified caspase 8 and RIPK3-independent cell killing pathway(s) is activated by TAK1 inhibition, which is another host defense mechanism. Our results demonstrate that apoptosis, pyroptosis, necroptosis together with unidentified pathways cooperatively act as the host defense in response to bacterial TAK1 inhibition. NIH R35GM139601.}, number={3}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Gonzalez-Calderon, Roland and Lopez-Perez, Wilfred and Sai, Kazuhito and Ninomiya-Tsuji, Jun}, year={2024}, month={Mar}, pages={S555–S555} }
 @article{nakanishi-hester_sai_ninomiya-tsuji_2024, title={The Mechanism and Roles of TAK1 hyperactivation in the Alzheimer's Disease Mouse Model}, volume={300}, ISSN={["1083-351X"]}, DOI={10.1016/j.jbc.2024.106732}, abstractNote={Neuroinflammation in the hippocampus is causally associated with Alzheimer's Disease (AD). However, the precise mechanisms of how inflammatory signaling in each cell type, neuron, oligodendrocyte, astrocyte, microglia, etc. impacts neuronal function remain elusive. Oligodendrocytes (OLGs), known for myelin formation and neuron support, emerge as potential contributors to chronic neuroinflammation. Unlike neurons, OLGs can regenerate throughout the lifespan, making them viable targets for understanding and potentially mitigating AD. We found that mitogen-activated protein kinase kinase kinase 7 (MAP3K7), also known as TAK1, a central player in intracellular inflammation signaling, is hyperactivated in the hippocampus of aged and AD model mice. We previously demonstrated that neuron lineage specific Tak1 gene deletion ameliorated neuron loss and cognitive impairment. These results suggest that TAK1-driven inflammatory signaling promotes AD. In our current study, my objectives are to elucidate the mechanism of TAK1 hyperactivation and its role, particularly, in OLGs. We found that compound stimulation of an inflammatory cytokine, TNF and a neurotransmitter glutamate-calcium influx activates TAK1. In addition, calcium calmodulin kinase II (CaMKII) is found to be involved in TAK1 activation by binding to and modulates TAK1 binding protein 2, TAB2. TAB2 serves a dual role in the regulation of TAK1 as it can recruit activators and deactivators of TAK1 and deleting Tab2 gene hyperactivates TAK1. These results collectively suggest that calcium-CaMKII-TAB2 blockade together with TNF hyperactivates TAK1. We are currently analyzing mice with OLG-specific Tab2 deletion (TAK1 hyper-activation). We found that Tab2 deletion blocks OLG differentiation, suggesting that TAK1 hyperactivation impairs OLG-derived support for neurons. Our results uncovered the importance of TAK1 in AD pathogenesis, offering potential avenues for therapeutic intervention. The study is funded by National Institute of Health, R35GM139601, 2019-AARG-NTF-641347.}, number={3}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Nakanishi-Hester, Aoi and Sai, Kazuhito and Ninomiya-Tsuji, Jun}, year={2024}, month={Mar}, pages={S556–S556} }
 @article{sai_nakanishi_scofield_tokarz_linder_cohen_ninomiya-tsuji_2023, title={Aberrantly activated TAK1 links neuroinflammation and neuronal loss in Alzheimer?s disease mouse models}, volume={136}, ISSN={["1477-9137"]}, url={https://doi.org/10.1242/jcs.260102}, DOI={10.1242/jcs.260102}, abstractNote={ABSTRACT
               Neuroinflammation is causally associated with Alzheimer's disease (AD) pathology. Reactive glia cells secrete various neurotoxic factors that impair neuronal homeostasis eventually leading to neuronal loss. Although the glial activation mechanism in AD has been relatively well studied, how it perturbs intraneuronal signaling, which ultimately leads to neuronal cell death, remains poorly understood. Here, we report that compound stimulation with the neurotoxic factors TNF and glutamate aberrantly activates neuronal TAK1 (also known as MAP3K7), which promotes the pathogenesis of AD in mouse models. Glutamate-induced Ca2+ influx shifts TNF signaling to hyper-activate TAK1 enzymatic activity through Ca2+/calmodulin-dependent protein kinase II, which leads to necroptotic cellular damage. Genetic ablation and pharmacological inhibition of TAK1 ameliorated AD-associated neuronal loss and cognitive impairment in the AD model mice. Our findings provide a molecular mechanism linking cytokines, Ca2+ signaling and neuronal necroptosis in AD.}, number={6}, journal={JOURNAL OF CELL SCIENCE}, publisher={The Company of Biologists}, author={Sai, Kazuhito and Nakanishi, Aoi and Scofield, Kimberly M. and Tokarz, Debra A. and Linder, Keith E. and Cohen, Todd J. and Ninomiya-Tsuji, Jun}, year={2023}, month={Mar} }
 @article{lopez-perez_sai_sakamachi_parsons_kathariou_ninomiya-tsuji_2021, title={TAK1 inhibition elicits mitochondrial ROS to block intracellular bacterial colonization}, volume={118}, ISSN={["0027-8424"]}, url={https://doi.org/10.1073/pnas.2023647118}, DOI={10.1073/pnas.2023647118}, abstractNote={Significance
          
            We have found that bacterial inhibition of host TAK1 inflammatory signaling elicits an alternative host defense mechanism involving production of mitochondrial reactive oxygen species through caspase 8 and RIPK3. This finding allows a reinterpretation of mouse phenotypes harboring tissue-specific gene deletion of
            Tak1
            , many of which die from tissue damage previously ascribed to impaired TAK1-dependent tissue homeostasis. We suggest that these phenotypes arise from misrecognition of compromised TAK1 as pathogen invasion.
          }, number={25}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, publisher={Proceedings of the National Academy of Sciences}, author={Lopez-Perez, Wilfred and Sai, Kazuhito and Sakamachi, Yosuke and Parsons, Cameron and Kathariou, Sophia and Ninomiya-Tsuji, Jun}, year={2021}, month={Jun} }
 @article{hsieh_agarwal_cholok_loder_kaneko_huber_chung_ranganathan_habbouche_li_et al._2019, title={Coordinating Tissue Regeneration Through Transforming Growth Factor-beta Activated Kinase 1 Inactivation and Reactivation}, volume={37}, ISSN={["1549-4918"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85062968911&partnerID=MN8TOARS}, DOI={10.1002/stem.2991}, abstractNote={Abstract
               Aberrant wound healing presents as inappropriate or insufficient tissue formation. Using a model of musculoskeletal injury, we demonstrate that loss of transforming growth factor-β activated kinase 1 (TAK1) signaling reduces inappropriate tissue formation (heterotopic ossification) through reduced cellular differentiation. Upon identifying increased proliferation with loss of TAK1 signaling, we considered a regenerative approach to address insufficient tissue production through coordinated inactivation of TAK1 to promote cellular proliferation, followed by reactivation to elicit differentiation and extracellular matrix production. Although the current regenerative medicine paradigm is centered on the effects of drug treatment (“drug on”), the impact of drug withdrawal (“drug off”) implicit in these regimens is unknown. Because current TAK1 inhibitors are unable to phenocopy genetic Tak1 loss, we introduce the dual-inducible COmbinational Sequential Inversion ENgineering (COSIEN) mouse model. The COSIEN mouse model, which allows us to study the response to targeted drug treatment (“drug on”) and subsequent withdrawal (“drug off”) through genetic modification, was used here to inactivate and reactivate Tak1 with the purpose of augmenting tissue regeneration in a calvarial defect model. Our study reveals the importance of both the “drug on” (Cre-mediated inactivation) and “drug off” (Flp-mediated reactivation) states during regenerative therapy using a mouse model with broad utility to study targeted therapies for disease. Stem Cells  2019;37:766–778
               
                  
               }, number={6}, journal={STEM CELLS}, author={Hsieh, Hsiao Hsin Sung and Agarwal, Shailesh and Cholok, David J. and Loder, Shawn J. and Kaneko, Kieko and Huber, Amanda and Chung, Michael T. and Ranganathan, Kavitha and Habbouche, Joe and Li, John and et al.}, year={2019}, month={Jun}, pages={766–778} }
 @article{sai_parsons_house_kathariou_ninomiya-tsuji_2019, title={Necroptosis mediators RIPK3 and MLKL suppress intracellular Listeria replication independently of host cell killing}, volume={218}, ISSN={["1540-8140"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85067213651&partnerID=MN8TOARS}, DOI={10.1083/jcb.201810014}, abstractNote={RIPK3, a key mediator of necroptosis, has been implicated in the host defense against viral infection primary in immune cells. However, gene expression analysis revealed that RIPK3 is abundantly expressed not only in immune organs but also in the gastrointestinal tract, particularly in the small intestine. We found that orally inoculated Listeria monocytogenes, a bacterial foodborne pathogen, efficiently spread and caused systemic infection in Ripk3-deficient mice while almost no dissemination was observed in wild-type mice. Listeria infection activated the RIPK3-MLKL pathway in cultured cells, which resulted in suppression of intracellular replication of Listeria. Surprisingly, Listeria infection–induced phosphorylation of MLKL did not result in host cell killing. We found that MLKL directly binds to Listeria and inhibits their replication in the cytosol. Our findings have revealed a novel functional role of the RIPK3-MLKL pathway in nonimmune cell-derived host defense against Listeria invasion, which is mediated through cell death–independent mechanisms.}, number={6}, journal={JOURNAL OF CELL BIOLOGY}, publisher={Rockefeller University Press}, author={Sai, Kazuhito and Parsons, Cameron and House, John S. and Kathariou, Sophia and Ninomiya-Tsuji, Jun}, year={2019}, month={Jun}, pages={1994–2005} }
 @article{liu_hayano_pan_inagaki_ninomiya-tsuji_sun_mishina_2018, title={Compound mutations in Bmpr1a and Tak1 synergize facial deformities via increased cell death}, volume={56}, ISSN={["1526-968X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85042327888&partnerID=MN8TOARS}, DOI={10.1002/dvg.23093}, abstractNote={SummaryBMP signaling plays a critical role in craniofacial development. Augmentation of BMPR1A signaling through neural crest‐specific expression of constitutively active Bmpr1a (caBmpr1a) results in craniofacial deformities in mice. To investigate whether deletion of Tak1 may rescue the craniofacial deformities caused by enhanced Smad‐dependent signaling through caBMPR1A, we generated embryos to activate transcription of caBmpr1a transgene and ablate Tak1 in neural crest derivatives at the same time. We found that deformities of the double mutant mice showed more severe than those with each single mutation, including median facial cleft and cleft palate. We found higher levels of cell death in the medial nasal and the lateral nasal processes at E10.5 in association with higher levels of p53 in the double mutant embryos. We also found higher levels of pSmad1/5/9 in the lateral nasal processes at E10.5 in the double mutant embryos. Western analyses revealed that double mutant embryos showed similar degrees of upregulation of pSmad1/5/9 with caBmpr1a or Tak1‐cKO embryos while the double mutant embryos showed higher levels of phospho‐p38 than caBmpr1a or Tak1‐cKO embryos at E17.5, but not at E10.5. It suggested that deletion of Tak1 aggravates the craniofacial deformities of the caBmpr1a mutants by increasing p53 and phospho‐p38 at different stage of embryogenesis.}, number={3}, journal={GENESIS}, author={Liu, Xia and Hayano, Satoru and Pan, Haichun and Inagaki, Maiko and Ninomiya-Tsuji, Jun and Sun, Hongchen and Mishina, Yuji}, year={2018}, month={Mar} }
 @article{mihaly_sakamachi_ninomiya-tsuji_morioka_2017, title={Erratum: Noncanonical cell death program independent of caspase activation cascade and necroptotic modules is elicited by loss of TGFβ-activated kinase 1}, volume={7}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/S41598-017-09609-Z}, DOI={10.1038/S41598-017-09609-Z}, abstractNote={A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.}, number={1}, journal={Scientific Reports}, publisher={Springer Nature}, author={Mihaly, September R. and Sakamachi, Yosuke and Ninomiya-Tsuji, Jun and Morioka, Sho}, year={2017}, month={Sep} }
 @article{mihaly_sakamachi_ninomiya-tsuji_morioka_2017, title={Noncanocial cell death program independent of caspase activation cascade and necroptotic modules is elicited by loss of TGF beta-activated kinase 1}, volume={7}, ISSN={["2045-2322"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85020443264&partnerID=MN8TOARS}, DOI={10.1038/s41598-017-03112-1}, abstractNote={AbstractProgrammed cell death (PCD) occurs in several forms including apoptosis and necroptosis. Apoptosis is executed by the activation of caspases, while necroptosis is dependent on the receptor interacting protein kinase 3 (RIPK3). Precise control of cell death is crucial for tissue homeostasis. Indeed, necroptosis is triggered by caspase inhibition to ensure cell death. Here we identified a previously uncharacterized cell death pathway regulated by TAK1, which is unexpectedly provoked by inhibition of caspase activity and necroptosis cascades. Ablation of TAK1 triggers spontaneous death in macrophages. Simultaneous inhibition of caspases and RIPK3 did not completely restore cell viability. Previous studies demonstrated that loss of TAK1 in fibroblasts causes TNF-induced apoptosis and that additional inhibition of caspase leads to necroptotic cell death. However, we surprisingly found that caspase and RIPK3 inhibitions do not completely suppress cell death in Tak1-deficient cells. Mechanistically, the execution of the third cell death pathway in Tak1-deficient macrophages and fibroblasts were mediated by RIPK1-dependent rapid accumulation of reactive oxygen species (ROS). Conversely, activation of RIPK1 was sufficient to induce cell death. Therefore, loss of TAK1 elicits noncanonical cell death which is mediated by RIPK1-induced oxidative stress upon caspase and necroptosis inhibition to further ensure induction of cell death.}, number={1}, journal={SCIENTIFIC REPORTS}, author={Mihaly, September R. and Sakamachi, Yosuke and Ninomiya-Tsuji, Jun and Morioka, Sho}, year={2017}, month={Jun} }
 @article{sakamachi_morioka_mihaly_takaesu_foley_fessler_ninomiya-tsuji_2017, title={TAK1 regulates resident macrophages by protecting lysosomal integrity}, volume={8}, ISSN={["2041-4889"]}, url={https://doi.org/10.1038/cddis.2017.23}, DOI={10.1038/cddis.2017.23}, abstractNote={AbstractHematopoietic cell survival and death is critical for development of a functional immune system. Here, we report that a protein kinase, TAK1, is selectively required for resident macrophage integrity during embryogenesis. Hematopoietic lineage-specific deletion of Tak1 gene (Tak1HKO) caused accumulation of cellular debris in the thymus in perinatal mice. Although no overt alteration in thymocytes and blood myeloid populations was observed in Tak1HKO mice, we found that thymic and lung macrophages were diminished. In the in vitro setting, Tak1 deficiency caused profound disruption of lysosomes and killed bone marrow-derived macrophages (BMDMs) without any exogenous stressors. Inhibition of the lysosomal protease, cathepsin B, partially blocked Tak1-deficient BMDM death, suggesting that leakage of the lysosomal contents is in part the cause of cell death. To identify the trigger of this cell death, we examined involvement of TNF and Toll-like receptor pathways. Among them, we found that deletion of Tnfr1 partially rescued cell death. Finally, we show that Tnfr1 deletion partially restored thymic and lung macrophages in vivo. These results suggest that autocrine and potentially paracrine TNF kills Tak1-deficient macrophages during development. Our results reveal that TAK1 signaling maintains proper macrophage populations through protecting lysosomal integrity.}, number={2}, journal={CELL DEATH & DISEASE}, author={Sakamachi, Yosuke and Morioka, Sho and Mihaly, September R. and Takaesu, Giichi and Foley, Julie F. and Fessler, Michael B. and Ninomiya-Tsuji, Jun}, year={2017}, month={Feb} }
 @article{hashimoto_simmons_kajino-sakamoto_tsuji_ninomiya-tsuji_2016, title={TAK1 Regulates the Nrf2 Antioxidant System Through Modulating p62/SQSTM1}, volume={25}, ISSN={["1557-7716"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85002932956&partnerID=MN8TOARS}, DOI={10.1089/ars.2016.6663}, abstractNote={AIMS
Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is the master transcriptional regulator of antioxidant gene expression. On increased oxidative stress, an adaptor for Nrf2 degradation, Kelch-like ECH-associated protein 1 (Keap1), is directly modulated by oxidants in the cytoplasm, which results in stabilization and activation of Nrf2. Nrf2 is also constitutively active, to some extent, in the absence of exogenous oxidative stress. We have previously demonstrated that intestinal epithelium-specific TGF-β-activated kinase 1 (TAK1) deletion downregulates the level of Nrf2 protein, resulting in an increase of reactive oxygen species (ROS) in a mouse model. We aim at determining the mechanism by which TAK1 modulates the level of Nrf2.


RESULTS
We found that TAK1 upregulated serine 351 phosphorylation of an autophagic adaptor protein, p62/Sequestosome-1 (SQSTM1), which facilitates interaction between p62/SQSTM1 and Keap1 and subsequent Keap1 degradation. This, ultimately, causes increased Nrf2. Tak1 deficiency reduced the phosphorylation of p62/SQSTM1, resulting in decreased steady-state levels of Nrf2 along with increased Keap1. We also found that this regulation is independent of the canonical redox-mediated Nrf2 activation mechanism. In Tak1-deficient intestinal epithelium, a synthetic phenolic electrophile, butylated hydroxyanisole still effectively upregulated Nrf2 and reduced ROS.


INNOVATION
Our results identify for the first time that TAK1 is a modulator of p62/SQSTM1-dependent Keap1 degradation and maintains the steady state-level of Nrf2.


CONCLUSION
TAK1 regulates Nrf2 through modulation of Keap-p62/SQSTM1 interaction. This regulation is important for homeostatic antioxidant protection in the intestinal epithelium. Antioxid. Redox Signal. 25, 953-964.}, number={17}, journal={ANTIOXIDANTS & REDOX SIGNALING}, author={Hashimoto, Kazunori and Simmons, Alicia N. and Kajino-Sakamoto, Rie and Tsuji, Yoshiaki and Ninomiya-Tsuji, Jun}, year={2016}, month={Dec}, pages={953–964} }
 @article{sai_morioka_takaesu_muthusamy_ghashghaei_hanafusa_matsumoto_ninomiya-tsuji_2016, title={TAK1 determines susceptibility to endoplasmic reticulum stress and leptin resistance in the hypothalamus}, volume={129}, ISSN={0021-9533 1477-9137}, url={http://dx.doi.org/10.1242/jcs.180505}, DOI={10.1242/jcs.180505}, abstractNote={Sustained endoplasmic reticulum (ER) stress disrupts normal cellular homeostasis and leads to the development of many types of human diseases including metabolic disorders. TAK1 is a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, and is activated by a diverse set of inflammatory stimuli. Here we demonstrate that TAK1 regulates ER stress and metabolic signaling through modulation of lipid biogenesis. We found that deletion of Tak1 increased ER volume and facilitated ER stress tolerance in cultured cells, which was mediated by upregulation of sterol-regulatory element binding proteins (SREBPs)-dependent lipogenesis. In the in vivo setting, central nervous system (CNS)-specific Tak1 deletion upregulated SREBP target lipogenic genes and blocked ER stress in the hypothalamus. Furthermore, CNS-specific Tak1 deletion prevented ER stress-induced hypothalamic leptin resistance and hyperphagic obesity under high fat diet (HFD). Thus, TAK1 is a critical regulator of ER stress in vivo, which could be a target for alleviation of ER stress and its associated disease conditions.}, number={9}, journal={Journal of Cell Science}, publisher={The Company of Biologists}, author={Sai, Kazuhito and Morioka, Sho and Takaesu, Giichi and Muthusamy, Nagendran and Ghashghaei, H. Troy and Hanafusa, Hiroshi and Matsumoto, Kunihiro and Ninomiya-Tsuji, Jun}, year={2016}, month={Mar}, pages={1855–1865} }
 @article{simmons_kajino-sakamoto_ninomiya-tsuji_2016, title={TAK1 regulates Paneth cell integrity partly through blocking necroptosis}, volume={7}, ISSN={["2041-4889"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85017564878&partnerID=MN8TOARS}, DOI={10.1038/cddis.2016.98}, abstractNote={AbstractPaneth cells reside at the base of crypts of the small intestine and secrete antimicrobial factors to control gut microbiota. Paneth cell loss is observed in the chronically inflamed intestine, which is often associated with increased reactive oxygen species (ROS). However, the relationship between Paneth cell loss and ROS is not yet clear. Intestinal epithelial-specific deletion of a protein kinase Tak1 depletes Paneth cells and highly upregulates ROS in the mouse model. We found that depletion of gut bacteria or myeloid differentiation factor 88 (Myd88), a mediator of bacteria-derived cell signaling, reduced ROS but did not block Paneth cell loss, suggesting that gut bacteria are the cause of ROS accumulation but bacteria-induced ROS are not the cause of Paneth cell loss. In contrast, deletion of the necroptotic cell death signaling intermediate, receptor-interacting protein kinase 3 (Ripk3), partially blocked Paneth cell loss. Thus, Tak1 deletion causes Paneth cell loss in part through necroptotic cell death. These results suggest that TAK1 participates in intestinal integrity through separately modulating bacteria-derived ROS and RIPK3-dependent Paneth cell loss.}, number={4}, journal={CELL DEATH & DISEASE}, author={Simmons, A. N. and Kajino-Sakamoto, R. and Ninomiya-Tsuji, J.}, year={2016}, month={Apr} }
 @article{morioka_sai_omori_ikeda_matsumoto_ninomiya-tsuji_2016, title={TAK1 regulates hepatic lipid homeostasis through SREBP}, volume={35}, ISSN={["1476-5594"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84961218902&partnerID=MN8TOARS}, DOI={10.1038/onc.2015.453}, abstractNote={Sterol-regulatory element-binding proteins (SREBPs) are key transcription factors regulating cholesterol and fatty acid biosynthesis. SREBP activity is tightly regulated to maintain lipid homeostasis, and is modulated upon extracellular stimuli such as growth factors. While the homeostatic SREBP regulation is well studied, stimuli-dependent regulatory mechanisms are still elusive. Here we demonstrate that SREBPs are regulated by a previously uncharacterized mechanism through transforming growth factor-β activated kinase 1 (TAK1), a signaling molecule of inflammation. We found that TAK1 binds to and inhibits mature forms of SREBPs. In an in vivo setting, hepatocyte-specific Tak1 deletion upregulates liver lipid deposition and lipogenic enzymes in the mouse model. Furthermore, hepatic Tak1 deficiency causes steatosis pathologies including elevated blood triglyceride and cholesterol levels, which are established risk factors for the development of hepatocellular carcinoma (HCC) and are indeed correlated with Tak1-deficiency-induced HCC development. Pharmacological inhibition of SREBPs alleviated the steatosis and reduced the expression level of the HCC marker gene in the Tak1-deficient liver. Thus, TAK1 regulation of SREBP critically contributes to the maintenance of liver homeostasis to prevent steatosis, which is a potentially important mechanism to prevent HCC development.}, number={29}, journal={ONCOGENE}, author={Morioka, S. and Sai, K. and Omori, E. and Ikeda, Y. and Matsumoto, K. and Ninomiya-Tsuji, J.}, year={2016}, month={Jul}, pages={3829–3838} }
 @article{mihaly_morioka_ninomiya-tsuji_takaesu_2014, title={Activated Macrophage Survival Is Coordinated by TAK1 Binding Proteins}, volume={9}, ISSN={["1932-6203"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84899697653&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0094982}, abstractNote={Macrophages play diverse roles in tissue homeostasis and immunity, and canonically activated macrophages are critically associated with acute inflammatory responses. It is known that activated macrophages undergo cell death after transient activation in some settings, and the viability of macrophages impacts on inflammatory status. Here we report that TGFβ- activated kinase (TAK1) activators, TAK1-binding protein 1 (TAB1) and TAK1-binding protein 2 (TAB2), are critical molecules in the regulation of activated macrophage survival. While deletion of Tak1 induced cell death in bone marrow derived macrophages even without activation, Tab1 or Tab2 deletion alone did not profoundly affect survival of naïve macrophages. However, in lipopolysaccharide (LPS)-activated macrophages, even single deletion of Tab1 or Tab2 resulted in macrophage death with both necrotic and apoptotic features. We show that TAB1 and TAB2 were redundantly involved in LPS-induced TAK1 activation in macrophages. These results demonstrate that TAK1 activity is the key to activated macrophage survival. Finally, in an in vivo setting, Tab1 deficiency impaired increase of peritoneal macrophages upon LPS challenge, suggesting that TAK1 complex regulation of macrophages may participate in in vivo macrophage homeostasis. Our results demonstrate that TAB1 and TAB2 are required for activated macrophages, making TAB1 and TAB2 effective targets to control inflammation by modulating macrophage survival.}, number={4}, journal={PLOS ONE}, author={Mihaly, September R. and Morioka, Sho and Ninomiya-Tsuji, Jun and Takaesu, Giichi}, year={2014}, month={Apr} }
 @article{ikeda_morioka_matsumoto_ninomiya-tsuji_2014, title={TAK1 Binding Protein 2 Is Essential for Liver Protection from Stressors}, volume={9}, ISSN={["1932-6203"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84894628175&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0088037}, abstractNote={The liver is the first line of defense from environmental stressors in that hepatocytes respond to and metabolize them. Hence, hepatocytes can be damaged by stressors. Protection against hepatic cell damage and cell death is important for liver function and homeostasis. TAK1 (MAP3K7) is an intermediate of stressors such as bacterial moieties–induced signal transduction pathways in several cell types. Tak1 deficiency has been reported to induce spontaneous hepatocellular carcinoma. However, the regulatory mechanism of TAK1 activity in liver stress response has not yet been defined. Here we report that activation of TAK1 through TAK1 binding protein 2 (TAB2) is required for liver protection from stressors. We found that a bacterial moiety, lipopolysaccharides (LPS), activated TAK1 in primary hepatocytes, which was diminished by deletion of TAB2. Mice having hepatocyte-specific deletion of the Tab2 gene exhibited only late-onset moderate liver lesions but were hypersensitive to LPS-induced liver injury. Furthermore, we show that a chemical stressor induced greatly exaggerated liver injury in hepatocyte-specific Tab2-deficient mice. These results demonstrate that TAB2 is a sensor of stress conditions in the liver and functions to protect the liver by activating the TAK1 pathway.}, number={2}, journal={PLOS ONE}, author={Ikeda, Yuka and Morioka, Sho and Matsumoto, Kunihiro and Ninomiya-Tsuji, Jun}, year={2014}, month={Feb} }
 @misc{mihaly_ninomiya-tsuji_morioka_2014, title={TAK1 control of cell death}, volume={21}, ISSN={["1476-5403"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84906072371&partnerID=MN8TOARS}, DOI={10.1038/cdd.2014.123}, abstractNote={Programmed cell death, a physiologic process for removing cells, is critically important in normal development and for elimination of damaged cells. Conversely, unattended cell death contributes to a variety of human disease pathogenesis. Thus, precise understanding of molecular mechanisms underlying control of cell death is important and relevant to public health. Recent studies emphasize that transforming growth factor-β-activated kinase 1 (TAK1) is a central regulator of cell death and is activated through a diverse set of intra- and extracellular stimuli. The physiologic importance of TAK1 and TAK1-binding proteins in cell survival and death has been demonstrated using a number of genetically engineered mice. These studies uncover an indispensable role of TAK1 and its binding proteins for maintenance of cell viability and tissue homeostasis in a variety of organs. TAK1 is known to control cell viability and inflammation through activating downstream effectors such as NF-κB and mitogen-activated protein kinases (MAPKs). It is also emerging that TAK1 regulates cell survival not solely through NF-κB but also through NF-κB-independent pathways such as oxidative stress and receptor-interacting protein kinase 1 (RIPK1) kinase activity-dependent pathway. Moreover, recent studies have identified TAK1's seemingly paradoxical role to induce programmed necrosis, also referred to as necroptosis. This review summarizes the consequences of TAK1 deficiency in different cell and tissue types from the perspective of cell death and also focuses on the mechanism by which TAK1 complex inhibits or promotes programmed cell death. This review serves to synthesize our current understanding of TAK1 in cell survival and death to identify promising directions for future research and TAK1's potential relevance to human disease pathogenesis.}, number={11}, journal={CELL DEATH AND DIFFERENTIATION}, author={Mihaly, S. R. and Ninomiya-Tsuji, J. and Morioka, S.}, year={2014}, month={Nov}, pages={1667–1676} }
 @article{morioka_broglie_omori_ikeda_takaesu_matsumoto_ninomiya-tsuji_2014, title={TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation}, volume={204}, ISSN={1540-8140 0021-9525}, url={http://dx.doi.org/10.1083/JCB.201305070}, DOI={10.1083/JCB.201305070}, abstractNote={TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8–mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)–dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.}, number={4}, journal={The Journal of Cell Biology}, publisher={Rockefeller University Press}, author={Morioka, Sho and Broglie, Peter and Omori, Emily and Ikeda, Yuka and Takaesu, Giichi and Matsumoto, Kunihiro and Ninomiya-Tsuji, Jun}, year={2014}, month={Feb}, pages={607–623} }
 @article{lane_yumoto_azhar_ninomiya-tsuji_inagaki_hu_deng_kim_mishina_kaartinen_2015, title={Tak1, Smad4 and Trim33 redundantly mediate TGF-beta 3 signaling during palate development}, volume={398}, ISSN={["1095-564X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84922712748&partnerID=MN8TOARS}, DOI={10.1016/j.ydbio.2014.12.006}, abstractNote={Transforming growth factor-beta3 (TGF-β3) plays a critical role in palatal epithelial cells by inducing palatal epithelial fusion, failure of which results in cleft palate, one of the most common birth defects in humans. Recent studies have shown that Smad-dependent and Smad-independent pathways work redundantly to transduce TGF-β3 signaling in palatal epithelial cells. However, detailed mechanisms by which this signaling is mediated still remain to be elucidated. Here we show that TGF-β activated kinase-1 (Tak1) and Smad4 interact genetically in palatal epithelial fusion. While simultaneous abrogation of both Tak1 and Smad4 in palatal epithelial cells resulted in characteristic defects in the anterior and posterior secondary palate, these phenotypes were less severe than those seen in the corresponding Tgfb3 mutants. Moreover, our results demonstrate that Trim33, a novel chromatin reader and regulator of TGF-β signaling, cooperates with Smad4 during palatogenesis. Unlike the epithelium-specific Smad4 mutants, epithelium-specific Tak1:Smad4- and Trim33:Smad4-double mutants display reduced expression of Mmp13 in palatal medial edge epithelial cells, suggesting that both of these redundant mechanisms are required for appropriate TGF-β signal transduction. Moreover, we show that inactivation of Tak1 in Trim33:Smad4 double conditional knockouts leads to the palatal phenotypes which are identical to those seen in epithelium-specific Tgfb3 mutants. To conclude, our data reveal added complexity in TGF-β signaling during palatogenesis and demonstrate that functionally redundant pathways involving Smad4, Tak1 and Trim33 regulate palatal epithelial fusion.}, number={2}, journal={DEVELOPMENTAL BIOLOGY}, author={Lane, Jamie and Yumoto, Kenji and Azhar, Mohamad and Ninomiya-Tsuji, Jun and Inagaki, Maiko and Hu, Yingling and Deng, Chu-Xia and Kim, Jieun and Mishina, Yuji and Kaartinen, Vesa}, year={2015}, month={Feb}, pages={231–241} }
 @article{moreno-garcia_sommer_rincon-arano_brault_ninomiya-tsuji_matesic_rawlings_2013, title={Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-kappa B Activation}, volume={33}, ISSN={["1098-5549"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84874701078&partnerID=MN8TOARS}, DOI={10.1128/mcb.06407-11}, abstractNote={ABSTRACT Antigen receptors activate pathways that control cell survival, proliferation, and differentiation. Two important targets of antigen receptors, NF-κB and Jun N-terminal kinase (JNK), are activated downstream of CARMA1, a scaffolding protein that nucleates a complex including BCL10, MALT1, and other IκB kinase (IKK)-signalosome components. Somatic mutations that constitutively activate CARMA1 occur frequently in diffuse large B cell lymphoma (DLBCL) and mediate essential survival signals. Mechanisms that downregulate this pathway might thus yield important therapeutic targets. Stimulation of antigen receptors induces not only BCL10 activation but also its degradation downstream of CARMA1, thereby ultimately limiting signals to its downstream targets. Here, using lymphocyte cell models, we identify a kinase-independent requirement for TAK1 and its adaptor, TAB1, in antigen receptor-induced BCL10 degradation. We show that TAK1 acts as an adaptor for E3 ubiquitin ligases that target BCL10 for degradation. Functionally, TAK1 overexpression restrains CARMA1-dependent activation of NF-κB by reducing BCL10 levels. TAK1 also promotes counterselection of NF-κB-addicted DLBCL lines by a dual mechanism involving kinase-independent degradation of BCL10 and kinase-dependent activation of JNK. Thus, by directly promoting BCL10 degradation, TAK1 counterbalances NF-κB and JNK signals essential for the activation and survival of lymphocytes and CARMA1-addicted lymphoma types.}, number={6}, journal={MOLECULAR AND CELLULAR BIOLOGY}, author={Moreno-Garcia, Miguel E. and Sommer, Karen and Rincon-Arano, Hector and Brault, Michelle and Ninomiya-Tsuji, Jun and Matesic, Lydia E. and Rawlings, David J.}, year={2013}, month={Mar}, pages={1149–1163} }
 @article{yumoto_thomas_lane_matsuzaki_inagaki_ninomiya-tsuji_scott_ray_ishii_maxson_et al._2013, title={TGF-beta-activated Kinase 1 (Tak1) Mediates Agonist-induced Smad Activation and Linker Region Phosphorylation in Embryonic Craniofacial Neural Crest-derived Cells}, volume={288}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84877705727&partnerID=MN8TOARS}, DOI={10.1074/jbc.m112.431775}, abstractNote={Background: The role of Smad-independent TGF-β signaling in craniofacial development is poorly elucidated. Results: In craniofacial mesenchymal cells, Tak1 regulates both R-Smad C-terminal and linker region phosphorylation in TGF-β signaling. Conclusion: Tak1 plays an irreplaceable role in craniofacial ecto-mesenchyme during embryogenesis. Significance: Understanding the mechanisms of TGF-β signaling contributes to knowledge of pathogenetic mechanisms underlying common craniofacial birth defects. Although the importance of TGF-β superfamily signaling in craniofacial growth and patterning is well established, the precise details of its signaling mechanisms are still poorly understood. This is in part because of the concentration of studies on the role of the Smad-dependent (so-called “canonical”) signaling pathways relative to the Smad-independent ones in many biological processes. Here, we have addressed the role of TGF-β-activated kinase 1 (Tak1, Map3k7), one of the key mediators of Smad-independent (noncanonical) TGF-β superfamily signaling in craniofacial development, by deleting Tak1 specifically in the neural crest lineage. Tak1-deficient mutants display a round skull, hypoplastic maxilla and mandible, and cleft palate resulting from a failure of palatal shelves to appropriately elevate and fuse. Our studies show that in neural crest-derived craniofacial ecto-mesenchymal cells, Tak1 is not only required for TGF-β- and bone morphogenetic protein-induced p38 Mapk activation but also plays a role in agonist-induced C-terminal and linker region phosphorylation of the receptor-mediated R-Smads. Specifically, we demonstrate that the agonist-induced linker region phosphorylation of Smad2 at Thr-220, which has been shown to be critical for full transcriptional activity of Smad2, is dependent on Tak1 activity and that in palatal mesenchymal cells TGFβRI and Tak1 kinases mediate both overlapping and distinct TGF-β2-induced transcriptional responses. To summarize, our results suggest that in neural crest-derived ecto-mesenchymal cells, Tak1 provides a critical point of intersection in a complex dialogue between the canonical and noncanonical arms of TGF-β superfamily signaling required for normal craniofacial development.}, number={19}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Yumoto, Kenji and Thomas, Penny S. and Lane, Jamie and Matsuzaki, Kouichi and Inagaki, Maiko and Ninomiya-Tsuji, Jun and Scott, Gregory J. and Ray, Manas K. and Ishii, Mamoru and Maxson, Robert and et al.}, year={2013}, month={May}, pages={13467–13480} }
 @article{omori_inagaki_mishina_matsumoto_ninomiya-tsuji_2012, title={Epithelial transforming growth factor  -activated kinase 1 (TAK1) is activated through two independent mechanisms and regulates reactive oxygen species}, volume={109}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.1116188109}, DOI={10.1073/pnas.1116188109}, abstractNote={Dysregulation in cellular redox systems results in accumulation of reactive oxygen species (ROS), which are causally associated with a number of disease conditions. Transforming growth factor β-activated kinase 1 (TAK1) is a signaling intermediate of innate immune signaling pathways and is critically involved in the redox regulation in vivo. Ablation of TAK1 causes accumulation of ROS, resulting in epithelial cell death and inflammation. Here we determine the mechanism by which TAK1 kinase is activated in epithelial tissues. TAB1 and TAB2 are structurally unrelated TAK1 binding protein partners. TAB2 is known to mediate polyubiquitin chain-dependent TAK1 activation in innate immune signaling pathways, whereas the role of TAB1 is not defined. We found that epithelial-specific TAB1 and TAB2 double- but not TAB1 or TAB2 single-knockout mice phenocopied epithelial-specific TAK1 knockout mice. We demonstrate that phosphorylation-dependent basal activity of TAK1 is dependent on TAB1. Ablation of both TAB1 and TAB2 diminished the activity of TAK1 in vivo and causes accumulation of ROS in the epithelial tissues. These results demonstrate that epithelial TAK1 activity is regulated through two unique, TAB1-dependent basal and TAB2-mediated stimuli-dependent mechanisms.}, number={9}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Omori, E. and Inagaki, M. and Mishina, Y. and Matsumoto, K. and Ninomiya-Tsuji, J.}, year={2012}, month={Feb}, pages={3365–3370} }
 @article{takaesu_inagaki_takubo_mishina_hess_dean_yoshimura_matsumoto_suda_ninomiya-tsuji_et al._2012, title={TAK1 (MAP3K7) Signaling Regulates Hematopoietic Stem Cells through TNF-Dependent and -Independent Mechanisms}, volume={7}, ISSN={1932-6203}, url={http://dx.doi.org/10.1371/journal.pone.0051073}, DOI={10.1371/journal.pone.0051073}, abstractNote={A cytokine/stress signaling kinase Tak1 (Map3k7) deficiency is known to impair hematopoietic progenitor cells. However, the role of TAK1 signaling in the stem cell function of the hematopoietic system is not yet well defined. Here we characterized hematopoietic stem cells (HSCs) harboring deletion of Tak1 and its activators, Tak1 binding proteins 1 and 2 (Tab1 and Tab2) using a competitive transplantation assay in a mouse model. Tak1 single or Tab1/Tab2 double deletions completely eliminated the reconstitution activity of HSCs, whereas Tab1 or Tab2 single deletion did not cause any abnormality. Tak1 single or Tab1/Tab2 double deficient lineage-negative, Sca-1+, c-Kit+ (LSK) cells did not proliferate and underwent cell death. We found that Tnfr1 deficiency restored the reconstitution activity of Tak1 deficient bone marrow cells for 6–18 weeks. However, the reconstitution activity of Tak1- and Tnfr1-double deficient bone marrow cells declined over the long term, and the number of phenotypically identified long-term hematopoietic stem cells were diminished. Our results indicate that TAB1- or TAB2-dependent activation of TAK1 is required for maintenance of the hematopoietic system through two mechanisms: one is prevention of TNF-dependent cell death and the other is TNF-independent maintenance of long-term HSC.}, number={11}, journal={PLoS ONE}, publisher={Public Library of Science (PLoS)}, author={Takaesu, Giichi and Inagaki, Maiko and Takubo, Keiyo and Mishina, Yuji and Hess, Paul R. and Dean, Gregg A. and Yoshimura, Akihiko and Matsumoto, Kunihiro and Suda, Toshio and Ninomiya-Tsuji, Jun and et al.}, editor={Tjwa, MarcEditor}, year={2012}, month={Nov}, pages={e51073} }
 @article{morioka_inagaki_komatsu_mishina_matsumoto_ninomiya-tsuji_2012, title={TAK1 kinase signaling regulates embryonic angiogenesis by modulating endothelial cell survival and migration}, volume={120}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84868609110&partnerID=MN8TOARS}, DOI={10.1182/blood-2012-03-416198}, number={18}, journal={Blood}, author={Morioka, S. and Inagaki, M. and Komatsu, Y. and Mishina, Y. and Matsumoto, K. and Ninomiya-Tsuji, J.}, year={2012}, pages={3846–3857} }
 @article{criollo_niso-santano_malik_michaud_morselli_marino_lachkar_arkhipenko_harper_pierron_et al._2011, title={Inhibition of autophagy by TAB2 and TAB3}, volume={30}, ISSN={["1460-2075"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-83555168258&partnerID=MN8TOARS}, DOI={10.1038/emboj.2011.413}, abstractNote={Article11 November 2011free access Inhibition of autophagy by TAB2 and TAB3 Alfredo Criollo Alfredo Criollo INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Mireia Niso-Santano Mireia Niso-Santano INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Shoaib Ahmad Malik Shoaib Ahmad Malik INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Mickael Michaud Mickael Michaud INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Eugenia Morselli Eugenia Morselli INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Guillermo Mariño Guillermo Mariño INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Sylvie Lachkar Sylvie Lachkar INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Alexander V Arkhipenko Alexander V Arkhipenko INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Search for more papers by this author Francis Harper Francis Harper Institut Gustave Roussy, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Gérard Pierron Gérard Pierron Institut Gustave Roussy, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Jean-Christophe Rain Jean-Christophe Rain Hybrigenics SA, Paris, France Search for more papers by this author Jun Ninomiya-Tsuji Jun Ninomiya-Tsuji Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC, USA Search for more papers by this author José M Fuentes José M Fuentes CIBERNED, Departamento de Bioquımica y Biologıa Molecular y Genética, EU Enfermería y TO, Universidad de Extremadura, Cacéres, Spain Search for more papers by this author Sergio Lavandero Sergio Lavandero Center Molecular Study of the Cell, Pharmaceutical and Chemical Science Faculty and Medicine Faculty, University of Chile, Santiago, Chile Cardiology Division, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Lorenzo Galluzzi Lorenzo Galluzzi INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Maria Chiara Maiuri Corresponding Author Maria Chiara Maiuri INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, FranceThese authors share senior co-authorship Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer INSERM, U848, Villejuif, France Metabolomics Platform, Institut Gustave Roussy, Villejuif, France Centre de Recherche des Cordeliers, Paris, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, FranceThese authors share senior co-authorship Search for more papers by this author Alfredo Criollo Alfredo Criollo INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Mireia Niso-Santano Mireia Niso-Santano INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Shoaib Ahmad Malik Shoaib Ahmad Malik INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Mickael Michaud Mickael Michaud INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Eugenia Morselli Eugenia Morselli INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Guillermo Mariño Guillermo Mariño INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Sylvie Lachkar Sylvie Lachkar INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Alexander V Arkhipenko Alexander V Arkhipenko INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Search for more papers by this author Francis Harper Francis Harper Institut Gustave Roussy, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Gérard Pierron Gérard Pierron Institut Gustave Roussy, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Jean-Christophe Rain Jean-Christophe Rain Hybrigenics SA, Paris, France Search for more papers by this author Jun Ninomiya-Tsuji Jun Ninomiya-Tsuji Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC, USA Search for more papers by this author José M Fuentes José M Fuentes CIBERNED, Departamento de Bioquımica y Biologıa Molecular y Genética, EU Enfermería y TO, Universidad de Extremadura, Cacéres, Spain Search for more papers by this author Sergio Lavandero Sergio Lavandero Center Molecular Study of the Cell, Pharmaceutical and Chemical Science Faculty and Medicine Faculty, University of Chile, Santiago, Chile Cardiology Division, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Lorenzo Galluzzi Lorenzo Galluzzi INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, France Search for more papers by this author Maria Chiara Maiuri Corresponding Author Maria Chiara Maiuri INSERM, U848, Villejuif, France Institut Gustave Roussy, Villejuif, France Université Paris Sud, Paris 11, Villejuif, FranceThese authors share senior co-authorship Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer INSERM, U848, Villejuif, France Metabolomics Platform, Institut Gustave Roussy, Villejuif, France Centre de Recherche des Cordeliers, Paris, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, FranceThese authors share senior co-authorship Search for more papers by this author Author Information Alfredo Criollo1,2,3,‡, Mireia Niso-Santano1,2,3,‡, Shoaib Ahmad Malik1,2,3, Mickael Michaud1,2,3, Eugenia Morselli1,2,3, Guillermo Mariño1,2,3, Sylvie Lachkar1,2,3, Alexander V Arkhipenko1,2, Francis Harper2,4, Gérard Pierron2,4, Jean-Christophe Rain5, Jun Ninomiya-Tsuji6, José M Fuentes7, Sergio Lavandero8,9, Lorenzo Galluzzi1,2,3, Maria Chiara Maiuri 1,2,3 and Guido Kroemer 1,10,11,12,13 1INSERM, U848, Villejuif, France 2Institut Gustave Roussy, Villejuif, France 3Université Paris Sud, Paris 11, Villejuif, France 4CNRS, UMR8122, Villejuif, France 5Hybrigenics SA, Paris, France 6Environmental and Molecular Toxicology, North Carolina State University, Raleigh, NC, USA 7CIBERNED, Departamento de Bioquımica y Biologıa Molecular y Genética, EU Enfermería y TO, Universidad de Extremadura, Cacéres, Spain 8Center Molecular Study of the Cell, Pharmaceutical and Chemical Science Faculty and Medicine Faculty, University of Chile, Santiago, Chile 9Cardiology Division, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA 10Metabolomics Platform, Institut Gustave Roussy, Villejuif, France 11Centre de Recherche des Cordeliers, Paris, France 12Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France 13Université Paris Descartes, Sorbonne Paris Cité, Paris, France ‡These authors contributed equally to this work *Corresponding authors: INSERM, U848, Institut Gustave Roussy, Pavillon de Recherche 1, 39 rue Camille Desmoulins, F–94805 Villejuif, France. Tel.: +33 1 4211 5216; Fax: +33 1 4211 6665; E-mail: [email protected] or Tel.: +33 1 4211 6046; Fax: +33 1 4211 6047; E-mail: [email protected] The EMBO Journal (2011)30:4908-4920https://doi.org/10.1038/emboj.2011.413 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Autophagic responses are coupled to the activation of the inhibitor of NF-κB kinase (IKK). Here, we report that the essential autophagy mediator Beclin 1 and TGFβ-activated kinase 1 (TAK1)-binding proteins 2 and 3 (TAB2 and TAB3), two upstream activators of the TAK1-IKK signalling axis, constitutively interact with each other via their coiled-coil domains (CCDs). Upon autophagy induction, TAB2 and TAB3 dissociate from Beclin 1 and bind TAK1. Moreover, overexpression of TAB2 and TAB3 suppresses, while their depletion triggers, autophagy. The expression of the C-terminal domain of TAB2 or TAB3 or that of the CCD of Beclin 1 competitively disrupts the interaction between endogenous Beclin 1, TAB2 and TAB3, hence stimulating autophagy through a pathway that requires endogenous Beclin 1, TAK1 and IKK to be optimally efficient. These results point to the existence of an autophagy-stimulatory ‘switch’ whereby TAB2 and TAB3 abandon inhibitory interactions with Beclin 1 to engage in a stimulatory liaison with TAK1. Introduction Macroautophagy (hereafter referred to as ‘autophagy’) is a catabolic pathway involving the sequestration of cytoplasmic material in double-membraned vesicles, the autophagosomes. Upon fusion with lysosomes, autophagosomes become autophagolysosomes and their content gets degraded by acidic hydrolases, allowing nutrients and macromolecules to fuel cellular metabolism or sustain stress responses (Klionsky, 2004; Mizushima et al, 2008). Multiple distinct perturbations of the cellular physiology can induce autophagy through a complex signalling network that crosstalks with several stress-response pathways (Kroemer et al, 2010; Green et al, 2011), including molecular cascades that are ignited by organellar damage as well as pathways leading to the activation of prominent transcription factors such as p53 (Tasdemir et al, 2008b; Scherz-Shouval et al, 2010) and NF-κB (Herrero-Martin et al, 2009; Criollo et al, 2010; Comb et al, 2011). Beclin 1 has been the first mammalian protein shown to play a critical role in the initiation of autophagy (Liang et al, 1999). Its complex interactome has a major influence on the positive and negative regulation of autophagy (Kang et al, 2011). One of the decisive events that ignites the autophagic machinery is the activation of the class III phosphatidylinositol 3-kinase (PI3KC3), also called VPS34, to generate phosphatidylinositol-3-phosphate, which is required for the initial steps of vesicle nucleation (Axe et al, 2008). Beclin 1 is an obligatory allosteric activator of VPS34, operating within the so-called ‘Beclin 1 core complex’ that involves Beclin 1, VPS15, VPS34 and, likely, AMBRA1 (He and Levine, 2010). Numerous additional proteins interact with Beclin 1. Pro-autophagic Beclin 1 interactors include ATG14 (also called ATG14L or BARKOR), UV radiation resistance-associated gene (UVRAG) and Bif-1/endophilin B1, which interacts with Beclin 1 via UVRAG. Autophagy-inhibitory interactors of Beclin 1 include RUN domain protein as Beclin 1 interacting and cysteine-rich containing (RUBICON), anti-apoptotic proteins from the BCL-2 family (BCL-2, BCL-XL and MCL-1) and the inositol-1,4,5 trisphosphate receptor, which interacts with Beclin 1 indirectly via BCL-2 (He and Levine, 2010; Kang et al, 2011). Beclin 1 possesses a BH3 domain (residues 114–123) through which it constitutively interacts with BCL-2-like proteins (Maiuri et al, 2007), and autophagy induction requires the dissociation of Beclin 1 from such inhibitory liaisons (He and Levine, 2010; Kang et al, 2011). The transcription factor NF-κB is activated when the inhibitor of NF-κB (IκB) is phosphorylated by the IκB kinase (IKK), a multiprotein complex that is composed by one regulatory (IKKγ, also known as NEMO) and two catalytic subunits (IKKα and IKKβ). IKK is activated in response to stressors as diverse as reactive oxygen species and DNA damage as well as by the ligation of death receptors (Baud and Karin, 2009). Frequently, the activation of IKK is mediated further upstream by yet another kinase, mitogen-activated protein kinase kinase kinase 7 (MAP3K7), better known as TGFβ-activated kinase 1 (TAK1) (Ninomiya-Tsuji et al, 1999; Takaesu et al, 2003; Landstrom, 2010). The phosphorylation of IκB by IKK stimulates IκB ubiquitination, thus targeting it for proteasomal degradation. In turn, IκB degradation allows NF-κB to translocate from the cytoplasm, where it is usually retained by IκB, to the nucleus, where NF-κB then becomes active as a cytoprotective and pro-inflammatory transcription factor (Baud and Karin, 2009). In both murine and human cells, the genetic inhibition of TAK1 or any of the IKK subunits (but not that of the NF-κB subunit p65) prevents the induction of autophagy in response to a panoply of different stimuli including starvation, rapamycin, p53 inhibition and endoplasmic reticulum stress (Herrero-Martin et al, 2009; Criollo et al, 2010; Comb et al, 2011). Conversely, constitutively active IKK subunits potently stimulate autophagy through a pathway that does not necessarily involve NF-κB, yet relies on the activation of AMPK and JNK1 (Criollo et al, 2010), as well as on several essential autophagy-related proteins including Beclin 1, ATG5 and LC3 (Comb et al, 2011). Altogether, these results point to the existence of a major crosstalk between autophagy and the TAK1-IKK signalling axis, yet do not reveal the molecular mechanisms through which these pathways intersect. Here, we report the discovery that TAK1-binding protein 2 (TAB2) and TAK1-binding protein 3 (TAB3) function as tonic inhibitors of autophagy. We found that TAB2 and TAB3 constitutively interact with Beclin 1 and that this liaison is lost upon treatment with physiological inducers of autophagy, causing TAB2 and TAB3 to bind TAK1 instead of Beclin 1. Competitive disruption of the Beclin 1/TAB2/TAB3 complex suffices to induce Beclin 1- and TAK1-dependent autophagy, underscoring the importance of the inhibitory interaction between Beclin 1, TAB2 and TAB3. Results Identification of TAB2 and TAB3 as novel Beclin 1 interactors To unveil a possible intersection between autophagy and the TAK1-IKK activation pathway, we identified Beclin 1 interactors in two yeast two-hybrid saturation screens based on a complex human random-primed cDNA library. This approach led us to identify 63 Beclin 1-interacting proteins. Beyond known interactors (such as UVRAG, ATG14 and ZWINT) (Behrends et al, 2010), our screens identified 11 new proteins that would bind Beclin 1 with an elevated Predicted Biological Score (Formstecher et al, 2005) (Figure 1A, also consultable at the following website http://pim.hybrigenics.com/). Among these 11 proteins, 1 (desmoplakin) has previously been described to bind multiple autophagy-relevant proteins including PIK3C3/VPS34 (but not Beclin 1), and two have been reported to interact with one single autophagy-related protein (PLEC1 with SQSTM1/p62 and SNX4 with RABGAP1) (Behrends et al, 2010). Of note, two among the putative Beclin 1 interactors, namely TAB2 and its close homologue TAB3, are known co-activators of TAK1 (Takaesu et al, 2000; Ishitani et al, 2003). Yeast two-hybrid technology allowed us to narrow down the domains that mediate the interaction of TAB2 and TAB3 with Beclin 1 to a C-terminal region that we called ‘Beclin-binding domain’ (BBD). The BBD spans from residues 526 to 657 in TAB2 and from residues 518 to 608 in TAB3 (Figure 1B). Co-immunoprecipitation experiments involving Beclin 1 and multiple TAB2 and TAB3 deletion constructs (Figure 1C) confirmed the interaction of full-length Beclin 1 with full-length TAB2 and TAB3, as well as with TAB2 and TAB3 fragments containing the C-terminal BBD. The binding of Beclin 1 to TAB2 and TAB3 was lost upon deletion of their BBDs (Figure 1D and E). Thus, TAB2 and TAB3 constitute novel bona fide members of the Beclin 1 interactome. Figure 1.Identification of novel Beclin 1 interactors. (A) Beclin 1 (BCN1) interactors identified by yeast two-hybrid technology. Proteins binding to BCN1 are listed and their interacting domains are indicated by black bars. Numbers refer to amino-acid positions. (B) Identification of TAB2 and TAB3 fragments interacting with BCN1 in the yeast two-hybrid system. Blue lines depict the fragments of TAB2 or TAB3 that were found to interact with BCN1 (numbers on the right refer to the amount of yeast clones identified for each fragment). The minimal domain required for the interaction is referred to as Beclin-binding domain (BBD). (C) Constructs derived from TAB2 and TAB3 used in this study. (D, E) Co-immunoprecipitation of TAB2 or TAB3 with BCN1. The indicated constructs, namely HA-tagged and T7-tagged TAB2, and TAB3 constructs in (D) and (E), respectively, and Flag-tagged Beclin 1 (Flag–BCN1) were transfected into HeLa cells alone or in combination. Twenty-four hours later, TAB2 and TAB3 were immunoprecipitated with antibodies specific for HA (D) or T7 (E) and the precipitate was separated by SDS–PAGE and revealed with an antibody specific for Flag. (F) Immunoprecipitation of endogenous BCN1 with endogenous TAB2 or TAB3. HeLa cells were subjected to autophagy induction with starvation conditions, 1 μM rapamycin or 30 μM pifithrin α (PFTα) for the indicated time and then processed for TAB2 or TAB3 immunoprecipitation followed by the immunodetection of BCN1, TAK1, TAB2 and TAB3. Results in (E) and (F) are representative for three independent experiments. Download figure Download PowerPoint TAB2 and TAB3 dissociate from Beclin 1 upon autophagy induction A His-tagged version of Beclin 1 was introduced together with epitope-tagged TAB2 or TAB3 into human cervical carcinoma HeLa cells, which were then driven into autophagy by culture in serum- and nutrient-free conditions (starvation), or by the administration of the mTOR inhibitor rapamycin or of the p53 inhibitor cyclic pifithrin α (PFTα). These three pro-autophagic stimuli all led to a decrease in the amount of TAB2 of TAB3 that co-immunoprecipitated with Beclin 1, as compared with control conditions (Supplementary Figure S1). Consistently, endogenous Beclin 1 co-immunoprecipitated with TAB2 or TAB3 (but not with TAK1) in control conditions, and this interaction was rapidly lost upon autophagy induction in HeLa cells (Figure 1F), as well as in other human cell lines (non small-cell lung cancer A549 cells, colorectal carcinoma HCT 116 cells) and mouse embryonic fibroblasts (MEFs, not shown). While in physiological conditions TAB2 and TAB3 failed to co-immunoprecipitate with TAK1, after the induction of autophagy with starvation, rapamycin or PFTα, both TAB2 and TAB3 were found to engage in such an interaction. Kinetic experiments revealed that TAB2 and TAB3 bind TAK1 as soon as they dissociate from Beclin 1 (Figure 1F). Inhibition of TAK1 by overexpression of a kinase-dead dominant-negative (DN) TAK1 mutant (TAK1K63W) (Ono et al, 2003; Figure 2A and B) or by means of a specific small-interfering RNA (siRNA) (Supplementary Figure S2) prevented the relocalization of a green fluorescence protein (GFP)–LC3 chimera from a diffuse pattern to discrete cytoplasmic puncta (Tasdemir et al, 2008a) by starvation, rapamycin or PFTα (Figure 2C and D). Similarly, TAK1 depletion prevented the autophagy-associated redistribution of a red fluorescent protein (RFP)-tagged FYVE domain (FYVE–RFP) (Zhang et al, 2007) to intracellular membranes containing phosphatidylinositol-3-phosphate (Figure 2E and F). Figure 2.Reduced interaction between Beclin 1, TAB and TAB3 in conditions of autophagy induction. (A, B) Inhibition of autophagy by dominant-negative (DN) TAK1. HeLa cells were co-transfected with a GFP–LC3-encoding construct plus pcDNA3.1 (empty vector), or plasmids for the expression of WT TAK1 (TAK1WT) or the DN TAK1K63W mutant. One day later, cells were either left untreated (control) or driven into autophagy by starvation or by the administration of 1 μM rapamycin or 30 μM pifithrin α (PFTα), followed by immunoblotting for the detection of TAK1 and endogenous LC3 (A) or immunofluorescence microscopy for the quantification of cells with cytosolic GFP–LC3 puncta (GFP–LC3VAC cells) (B) (mean values±s.d., n=3; *P<0.01 versus control cells). GAPDH levels were monitored to ensure equal loading. (C, D) Inhibition of autophagy by knockdown of VPS34, Beclin 1 (BCN1) and TAK1. siRNAs that effectively deplete VPS34, BCN1 and TAK1 were co-transfected with a GFP–LC3-encoding plasmid in HeLa cells. Autophagy was then induced as in (A) and the frequency of GFP–LC3VAC cells (mean values±s.d., n=3; *P<0.01 versus control cells) was determined. (E, F) The same setting shown in (C, D) was performed with U2OS cells and FYVE–RFP (mean values±s.d., n=3; *P<0.01, **P<0.001 versus control cells). Download figure Download PowerPoint Altogether, these results support the idea that TAB2 and TAB3 dissociate from Beclin 1 to engage with TAK1 when autophagy is induced. TAB2 and TAB3 are endogenous inhibitors of autophagy Depletion of TAB2 or TAB3 by specific siRNAs induced the accumulation of GFP–LC3+ dots in HeLa and human osteosarcoma U2OS cells stably expressing GFP–LC3 (Figure 3A and B), in HeLa cells transiently transfected with a GFP–LC3-encoding plasmid as well as in HCT 116 cells (Supplementary Figure S3). Moreover, TAB2 or TAB3 knockdown stimulated the lipidation of endogenous LC3, an autophagy-associated post-translational modification that enhances its electrophoretic mobility (shift from the LC3-I to the LC3-II form), and reduced the abundance of the autophagic substrate SQSTM1/p62 (Figure 3C), suggesting that TAB2 and TAB3 act as endogenous inhibitors of autophagy. Accordingly, the depletion of TAB2 or TAB3 led to the accumulation of bona fide autophagosomes and autophagolysosomes, as determined by transmission electron microscopy (Figure 3D and E). Epistatic experiments revealed that the simultaneous knockdown of TAB2 and TAB3 induced only slightly more GFP–LC3+ puncta than the depletion of each of these proteins alone. Moreover, the induction of autophagy by TAB2 depletion was inhibited by transfection of non-interferable TAB2 or wild-type (WT) TAB3 (and vice versa TAB2 transfection antagonized autophagy induction by TAB3 depletion), suggesting that both proteins inhibit the formation of autophagic puncta in a similar, overlapping manner (Figure 3F). Moreover, the depletion of either TAB induced the formation of FYVE–RFP+ puncta to similar extents (Figure 3G), suggesting that both TAB2 and TAB3 usually restrain the lipid kinase activity of the Beclin 1 complex. Figure 3.Induction of autophagosomes by depletion of TAB2 or TAB3. (A, B) Detection of autophagic GFP–LC3+ puncta. HeLa or U2OS cells stably expressing GFP–LC3 were transfected with siRNAs targeting TAK1, TAB1, TAB2 or TAB3 or with a control siRNA (siUNR). One day later, the subcellular localization and abundance of GFP–LC3 or immunostained TAB2 or TAB3 was determined by epifluorescence microscopy. Representative images are shown in (A) (HeLa cells) and quantitative results (mean values±s.d., n=3; *P<0.01 versus siUNR-transfected cells) are depicted in (B) (U2OS cells). (C) Lipidation of LC3 induced by TAB2 or TAB3 knockdown. Representative immunoblots showing the conversion of non-lipidated LC3 (LC3-I) to its lipidated variant (LC3-II) as well as SQSTM1/p62 protein levels are shown. GAPDH levels were monitored to ensure equal loading. (D, E) Quantification of autophagosomes and autophagolysosomes by transmission electron microscopy. Representative images of HeLa cells transfected with siUNR or with TAB2- or TAB3-targeting siRNAs are shown in (D), and quantitative results are depicted in (E) (mean values±s.d., n=3; *P<0.01 versus siUNR-transfected cells). (F) Epistatic analysis of the effects of TAB2 and TAB3 depletion on autophagy. HeLa cells stably expressing GFP–LC3 were transfected with siRNAs specific for TAB2 or TAB3 and/or with cDNAs coding for full-length HA-tagged TAB2 (HA–TAB2) or TAB3 (HA–TAB3). Twenty-four hours later, the frequency of cells exhibiting >5 GFP–LC3+ cytosolic puncta (GFP–LC3VAC cells) was determined. Results are mean values±s.d. (n=3; *P<0.01 versus siUNR-transfected cells). (G) U2OS cells stably expressing FYVE–RFP were transfected with siUNR, or with siRNAs specific for TAB2, TAB3, VPS34, Beclin 1 (BCN1) and TAK1, in the indicated combinations. Forty-eight hours later, the percentage of cells with RFP–FYVE+ puncta cells was determined. Results are mean values±s.d. (n=3; *P<0.01 versus siUNR-transfected cells). Download figure Download PowerPoint The accumulation of autophagosomes may result from enhanced sequestration of cytoplasmic material (increased on-rate) as well as from reduced removal of autophagosomes by fusion with lysosomes (reduced off-rate). Therefore, we measured autophagosome formation induced by TAB depletion in the absence or presence of bafilomycin A1 (BafA1), which inhibited the colocalization of the autophagic marker GFP–LC3 and the lysosomal marker LAMP2, in accord with its known capacity to block the autophagosome–lysosome fusion (Mizushima et al, 2010) (Figure 4A–C). In the presence of BafA1, the depletion of TAB2 and TAB3 resulted in more GFP–LC3+ puncta than in its absence (Figure 4D). Similar flux determinations were performed in the presence of an alternative lysosomal inhibitor, ammonium chloride, or protease inhibitors (E64d plus pepstatin A) (Supplementary Figure S4). Also, MEFs lacking TAB2 expression due to homologous recombination (Tab2−/−), but neither WT MEFs nor their Tab1−/− counterparts, manifested increased LC3 lipidation, both in the absence and in the presence of BafA1 (Figure 4E). Autophagy induced by TAB2 or TAB3 knockdown followed the canonical pathway, as it was reduced upon depletion of essential autophagy proteins such as Beclin 1, its associated phosphatidylinositol 3-kinase VPS34, ATG5 and ATG7 (Figure 4F). Moreover, it involved the obligatory contribution of TAK1 and that of the three subunits of the IKK complex, as shown by additional experiments of siRNA-mediated knockdown (Figure 4G) or transfection with DN TAK1 (Figure 4H). In conclusion, TAB2 and TAB3 are endogenous inhibitors of the canonical autophagic pathway, which requires the action of kinases from the TAK1-IKK signalling axis. Figure 4.Mechanisms of autophagy induced by depletion of TAB2 or TAB3. (A–D) Impact of bafilomycin A1 (BafA1) on the induction of GFP–LC3+ puncta by TAB2 and TAB3 depletion. HeLa cells stably expressing GFP–LC3 were transfected with a control siRNA (siUNR) or with siRNAs targeting TAB2 and TAB3 for 24 h. During the last 12 h of this period, BafA1 was optionally added. After fixation and permeabilization, LAMP2 was detected by immunofluorescence. Representative confocal microphotographs for the TAB2 siRNA are shown (A), together with the profiles of colocalization of fluorescent signals (B) along the indicated direction (α–ω). Columns in (C) represent the percentage of colocalization of GFP–LC3 and LAMP2 (mean values±s.d.; *P<0.01 versus siUNR-transfected cells), as quantified in at least 50 cells for each condition. The frequency (mean±s.d.) of cells with >5 GFP–LC3+ cytosolic puncta (GFP–LC3VAC cells) is plotted in (D). (E) Impact of BafA1 on LC3 lipidation. MEFs with the indicated genotypes were cultured in complete medium supplemented with BafA1 for 12 h and the proportion of LC3-I/LC3-II was determined by immunoblotting. GAPDH levels were monitored to ensure equal loading. (F, G) Impact of autophagy-relevant proteins and of the TAK1-IKK signalling axis on GFP–LC3 aggregation induced by the depletion of TAB2 or TAB3. HeLa cells stably expressing GFP–LC3 were transfected with siUNR or with siRNAs targeting the indicated proteins, alone or in combination, and 48 h later GFP–LC3VAC cells were quantified (mean values±s.d., n=4; *P<0.01 versus siUNR-transfected cells). (H) Inhibition of autophagy by dominant-negative (DN) TAK1. HeLa cells stably expressing GFP–LC3 were co-transfected with pcDNA3.1 (empty vector) or with plasmids encoding WT (TAK1WT) or a DN TAK1 variant (TAK1K63W) together with the indicated siRNAs for 24 h, followed by the quantification of GFP–LC3VAC cells (mean values±s.d., n=3, *P<0.01 versus siUNR-, pcDNA3.1-transfected cells). Download figure Download PowerPoint Dissociation of TAB2 and TAB3 from Beclin 1 induces autophagy Overexpression of full-length TAB2 or TAB3 inhibited starvation-, rapamycin-}, number={24}, journal={EMBO JOURNAL}, author={Criollo, Alfredo and Niso-Santano, Mireia and Malik, Shoaib Ahmad and Michaud, Mickael and Morselli, Eugenia and Marino, Guillermo and Lachkar, Sylvie and Arkhipenko, Alexander V. and Harper, Francis and Pierron, Gerard and et al.}, year={2011}, month={Dec}, pages={4908–4920} }
 @article{omori_matsumoto_ninomiya-tsuji_2011, title={Non-canonical beta-catenin degradation mediates reactive oxygen species-induced epidermal cell death}, volume={30}, ISSN={["0950-9232"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-79960836358&partnerID=MN8TOARS}, DOI={10.1038/onc.2011.49}, abstractNote={β-Catenin is constantly degraded through the ubiquitin-proteasomal pathway. In this study, we report that a different type of β-catenin degradation is causally involved in epidermal cell death. We observed that reactive oxygen species (ROS) caused β-catenin degradation in the epidermal cells through a caspase-dependent mechanism, which results in disruption of cell adhesion. Disruption of cell adhesion increased ROS and activated caspases. Upregulation of the intact β-catenin blocked ROS accumulation and caspase activation. These results indicate that a feed-forward loop consisting of ROS, caspases activation and β-catenin degradation induces epidermal cell death.}, number={30}, journal={ONCOGENE}, author={Omori, E. and Matsumoto, K. and Ninomiya-Tsuji, J.}, year={2011}, month={Jul}, pages={3336–3344} }
 @article{omori_matsumoto_zhu_smart_ninomiya-tsuji_2010, title={Ablation of TAK1 Upregulates Reactive Oxygen Species and Selectively Kills Tumor Cells}, volume={70}, ISSN={0008-5472 1538-7445}, url={http://dx.doi.org/10.1158/0008-5472.can-10-1227}, DOI={10.1158/0008-5472.can-10-1227}, abstractNote={Abstract
               TAK1 kinase activates multiple transcription factors and regulates the level of reactive oxygen species (ROS). We have previously reported that ablation of TAK1 in keratinocytes causes hypersensitivity to ROS-induced cell apoptosis. It is known that some tumor cells produce ROS at higher levels compared with normal cells. We used inducible epidermal-specific TAK1 knockout mice and examined whether ablation of TAK1 in preexisting skin tumors could cause an increase in ROS and result in tumor cell death. Deletion of tak1 gene in skin tumors caused the accumulation of ROS and increased apoptosis, and skin tumors totally regressed within 5 to 10 days. Normal skin did not exhibit any significant abnormality on tak1 gene deletion. Thus, TAK1 kinase could be a new and effective molecular target for ROS-based tumor killing. Cancer Res; 70(21); 8417–25. ©2010 AACR.}, number={21}, journal={Cancer Research}, publisher={American Association for Cancer Research (AACR)}, author={Omori, Emily and Matsumoto, Kunihiro and Zhu, Songyun and Smart, Robert C. and Ninomiya-Tsuji, Jun}, year={2010}, month={Oct}, pages={8417–8425} }
 @article{sakamoto_huang_iwasaki_hailemariam_ninomiya-tsuji_tsuji_2010, title={Regulation of Genotoxic Stress Response by Homeodomain-interacting Protein Kinase 2 through Phosphorylation of Cyclic AMP Response Element-binding Protein at Serine 271}, volume={21}, ISSN={["1939-4586"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77955599626&partnerID=MN8TOARS}, DOI={10.1091/mbc.e10-01-0015}, abstractNote={ CREB (cyclic AMP response element-binding protein) is a stimulus-induced transcription factor that plays pivotal roles in cell survival and proliferation. The transactivation function of CREB is primarily regulated through Ser-133 phosphorylation by cAMP-dependent protein kinase A (PKA) and related kinases. Here we found that homeodomain-interacting protein kinase 2 (HIPK2), a DNA-damage responsive nuclear kinase, is a new CREB kinase for phosphorylation at Ser-271 but not Ser-133, and activates CREB transactivation function including brain-derived neurotrophic factor (BDNF) mRNA expression. Ser-271 to Glu-271 substitution potentiated the CREB transactivation function. ChIP assays in SH-SY5Y neuroblastoma cells demonstrated that CREB Ser-271 phosphorylation by HIPK2 increased recruitment of a transcriptional coactivator CBP (CREB binding protein) without modulation of CREB binding to the BDNF CRE sequence. HIPK2−/− MEF cells were more susceptible to apoptosis induced by etoposide, a DNA-damaging agent, than HIPK2+/+ cells. Etoposide activated CRE-dependent transcription in HIPK2+/+ MEF cells but not in HIPK2−/− cells. HIPK2 knockdown in SH-SY5Y cells decreased etoposide-induced BDNF mRNA expression. These results demonstrate that HIPK2 is a new CREB kinase that regulates CREB-dependent transcription in genotoxic stress. }, number={16}, journal={MOLECULAR BIOLOGY OF THE CELL}, author={Sakamoto, Kensuke and Huang, Bo-Wen and Iwasaki, Kenta and Hailemariam, Kiros and Ninomiya-Tsuji, Jun and Tsuji, Yoshiaki}, year={2010}, month={Aug}, pages={2966–2974} }
 @article{kajino-sakamoto_omori_nighot_blikslager_matsumoto_ninomiya-tsuji_2010, title={TGF-β–Activated Kinase 1 Signaling Maintains Intestinal Integrity by Preventing Accumulation of Reactive Oxygen Species in the Intestinal Epithelium}, volume={185}, ISSN={0022-1767 1550-6606}, url={http://dx.doi.org/10.4049/jimmunol.0903587}, DOI={10.4049/jimmunol.0903587}, abstractNote={Abstract
               The intestinal epithelium is constantly exposed to inducers of reactive oxygen species (ROS), such as commensal microorganisms. Levels of ROS are normally maintained at nontoxic levels, but dysregulation of ROS is involved in intestinal inflammatory diseases. In this article, we report that TGF-β–activated kinase 1 (TAK1) is a key regulator of ROS in the intestinal epithelium. tak1 gene deletion in the mouse intestinal epithelium caused tissue damage involving enterocyte apoptosis, disruption of tight junctions, and inflammation. Disruption of TNF signaling, which is a major intestinal damage inducer, rescued the inflammatory conditions but not apoptosis or disruption of tight junctions in the TAK1-deficient intestinal epithelium, suggesting that TNF is not a primary inducer of the damage noted in TAK1-deficient intestinal epithelium. We found that TAK1 deficiency resulted in reduced expression of several antioxidant-responsive genes and reduced the protein level of a key antioxidant transcription factor NF-E2–related factor 2, which resulted in accumulation of ROS. Exogenous antioxidant treatment reduced apoptosis and disruption of tight junctions in the TAK1-deficient intestinal epithelium. Thus, TAK1 signaling regulates ROS through transcription factor NF-E2–related factor 2, which is important for intestinal epithelial integrity.}, number={8}, journal={The Journal of Immunology}, publisher={The American Association of Immunologists}, author={Kajino-Sakamoto, Rie and Omori, Emily and Nighot, Prashant K. and Blikslager, Anthony T. and Matsumoto, Kunihiro and Ninomiya-Tsuji, Jun}, year={2010}, month={Sep}, pages={4729–4737} }
 @article{kim_kajino-sakamoto_omori_jobin_ninomiya-tsuji_2009, title={Intestinal Epithelial-Derived TAK1 Signaling Is Essential for Cytoprotection against Chemical-Induced Colitis}, volume={4}, ISSN={["1932-6203"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84887212416&partnerID=MN8TOARS}, DOI={10.1371/journal.pone.0004561}, abstractNote={Background We have previously reported that intestinal epithelium-specific TAK1 deleted mice exhibit severe inflammation and mortality at postnatal day 1 due to TNF-induced epithelial cell death. Although deletion of TNF receptor 1 (TNFR1) can largely rescue those neonatal phenotypes, mice harboring double deletion of TNF receptor 1 (TNFR1) and intestinal epithelium-specific deletion of TAK1 (TNFR1KO/TAK1IEKO) still occasionally show increased inflammation. This indicates that TAK1 is important for TNF-independent regulation of intestinal integrity. Methodology/Principal Findings In this study, we investigated the TNF-independent role of TAK1 in the intestinal epithelium. Because the inflammatory conditions were sporadically developed in the double mutant TNFR1KO/TAK1IEKO mice, we hypothesize that epithelial TAK1 signaling is important for preventing stress-induced barrier dysfunction. To test this hypothesis, the TNFR1KO/TAK1IEKO mice were subjected to acute colitis by administration of dextran sulfate sodium (DSS). We found that loss of TAK1 significantly augments DSS-induced experimental colitis. DSS induced weight loss, intestinal damages and inflammatory markers in TNFR1KO/TAK1IEKO mice at higher levels compared to the TNFR1KO control mice. Apoptosis was strongly induced and epithelial cell proliferation was decreased in the TAK1-deficient intestinal epithelium upon DSS exposure. These suggest that epithelial-derived TAK1 signaling is important for cytoprotection and repair against injury. Finally, we showed that TAK1 is essential for interleukin 1- and bacterial components-induced expression of cytoprotective factors such as interleukin 6 and cycloxygenase 2. Conclusions Homeostatic cytokines and microbes-induced intestinal epithelial TAK1 signaling regulates cytoprotective factors and cell proliferation, which is pivotal for protecting the intestinal epithelium against injury.}, number={2}, journal={PLOS ONE}, author={Kim, Jae-Young and Kajino-Sakamoto, Rie and Omori, Emily and Jobin, Christian and Ninomiya-Tsuji, Jun}, year={2009}, month={Feb} }
 @article{morioka_omori_kajino_kajino-sakamoto_matsumoto_ninomiya-tsuji_2009, title={TAK1 kinase determines TRAIL sensitivity by modulating reactive oxygen species and cIAP}, volume={28}, ISSN={["1476-5594"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-67349205025&partnerID=MN8TOARS}, DOI={10.1038/onc.2009.110}, abstractNote={TNF-related apoptosis-inducing ligand (TRAIL) is a potent inducer of cell death in several cancer cells, but many cells are resistant to TRAIL. The mechanism that determines sensitivity to TRAIL-killing is still elusive. Here we report that deletion of TAK1 kinase greatly increased activation of caspase-3 and cell death after TRAIL stimulation in keratinocytes, fibroblasts and cancer cells. Although TAK1 kinase is involved in NF-kappaB pathway, ablation of NF-kappaB did not alter sensitivity to TRAIL. We found that TRAIL could induce accumulation of reactive oxygen species (ROS) when TAK1 was deleted. Furthermore, we found that TAK1 deletion induced TRAIL-dependent downregulation of cIAP, which enhanced activation of caspase-3. These results show that TAK1 deletion facilitates TRAIL-induced cell death by activating caspase through ROS and downregulation of cIAP. Thus, inhibition of TAK1 can be an effective approach to increase TRAIL sensitivity.}, number={23}, journal={ONCOGENE}, author={Morioka, S. and Omori, E. and Kajino, T. and Kajino-Sakamoto, R. and Matsumoto, K. and Ninomiya-Tsuji, J.}, year={2009}, month={Jun}, pages={2257–2265} }
 @article{broglie_matsumoto_akira_brautigan_ninomiya-tsuji_2010, title={Transforming Growth Factor beta-activated Kinase 1 (TAK1) Kinase Adaptor, TAK1-binding Protein 2, Plays Dual Roles in TAK1 Signaling by Recruiting Both an Activator and an Inhibitor of TAK1 Kinase in Tumor Necrosis Factor Signaling Pathway}, volume={285}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-77449125293&partnerID=MN8TOARS}, DOI={10.1074/jbc.M109.090522}, abstractNote={Transforming growth factor β-activated kinase 1 (TAK1) kinase is an indispensable signaling intermediate in tumor necrosis factor (TNF), interleukin 1, and Toll-like receptor signaling pathways. TAK1-binding protein 2 (TAB2) and its closely related protein, TAB3, are binding partners of TAK1 and have previously been identified as adaptors of TAK1 that recruit TAK1 to a TNF receptor signaling complex. TAB2 and TAB3 redundantly mediate activation of TAK1. In this study, we investigated the role of TAB2 by analyzing fibroblasts having targeted deletion of the tab2 gene. In TAB2-deficient fibroblasts, TAK1 was associated with TAB3 and was activated following TNF stimulation. However, TAB2-deficient fibroblasts displayed a significantly prolonged activation of TAK1 compared with wild type control cells. This suggests that TAB2 mediates deactivation of TAK1. We found that a TAK1-negative regulator, protein phosphatase 6 (PP6), was recruited to the TAK1 complex in wild type but not in TAB2-deficient fibroblasts. Furthermore, we demonstrated that both PP6 and TAB2 interacted with the polyubiquitin chains and this interaction mediated the assembly with TAK1. Our results indicate that TAB2 not only activates TAK1 but also plays an essential role in the deactivation of TAK1 by recruiting PP6 through a polyubiquitin chain-dependent mechanism.}, number={4}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Broglie, Peter and Matsumoto, Kunihiro and Akira, Shizuo and Brautigan, David L. and Ninomiya-Tsuji, Jun}, year={2010}, month={Jan}, pages={2333–2339} }
 @article{kajino-sakamoto_inagaki_kim_robine_matsumoto_jobin_ninomiya-tsuji_2008, title={203 TAK1 Is Essential for Intestinal Epithelial Cell Survival and Regulates Intestinal Integrity}, volume={134}, ISSN={0016-5085}, url={http://dx.doi.org/10.1016/S0016-5085(08)60172-9}, DOI={10.1016/S0016-5085(08)60172-9}, number={4}, journal={Gastroenterology}, publisher={Elsevier BV}, author={Kajino-Sakamoto, Rie and Inagaki, Maiko and Kim, Jae-Young and Robine, Sylvie and Matsumoto, Kunihiro and Jobin, Christian and Ninomiya-Tsuji, Jun}, year={2008}, month={Apr}, pages={A-35-A-36} }
 @article{kajino-sakamoto_inagaki_lippert_akira_robine_matsumoto_jobin_ninomiya-tsuji_2008, title={Enterocyte-derived TAK1 signaling prevents epithelium apoptosis and the development of ileitis and colitis}, volume={181}, ISSN={["1550-6606"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-49049095991&partnerID=MN8TOARS}, DOI={10.4049/jimmunol.181.2.1143}, abstractNote={Abstract
               Recent studies have revealed that TAK1 kinase is an essential intermediate in several innate immune signaling pathways. In this study, we investigated the role of TAK1 signaling in maintaining intestinal homeostasis by generating enterocyte-specific constitutive and inducible gene-deleted TAK1 mice. We found that enterocyte-specific constitutive TAK1-deleted mice spontaneously developed intestinal inflammation as observed by histological analysis and enhanced expression of IL-1β, MIP-2, and IL-6 around the time of birth, which was accompanied by significant enterocyte apoptosis. When TAK1 was deleted in the intestinal epithelium of 4-wk-old mice using an inducible knockout system, enterocytes underwent apoptosis and intestinal inflammation developed within 2–3 days following the initiation of gene deletion. We found that enterocyte apoptosis and intestinal inflammation were strongly attenuated when enterocyte-specific constitutive TAK1-deleted mice were crossed to TNF receptor 1−/− mice. However, these mice later (>14 days) developed ileitis and colitis. Thus, TAK1 signaling in enterocytes is essential for preventing TNF-dependent epithelium apoptosis and the TNF-independent development of ileitis and colitis. We propose that aberration in TAK1 signaling might disrupt intestinal homeostasis and favor the development of inflammatory disease.}, number={2}, journal={JOURNAL OF IMMUNOLOGY}, author={Kajino-Sakamoto, Rie and Inagaki, Maiko and Lippert, Elisabeth and Akira, Shizuo and Robine, Sylvie and Matsumoto, Kunihiro and Jobin, Christian and Ninomiya-Tsuji, Jun}, year={2008}, month={Jul}, pages={1143–1152} }
 @article{inagaki_komatsu_scott_yamada_ray_ninomiya-tsuji_mishina_2008, title={Generation of a conditional mutant allele for Tab1 in mouse}, volume={46}, ISSN={["1526-968X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-52049099157&partnerID=MN8TOARS}, DOI={10.1002/dvg.20418}, abstractNote={AbstractTAK1 binding protein 1 (TAB1) binds and induces autophosphorylation of TGF‐β activating kinase (TAK1). TAK1, a mitogen‐activated kinase kinase kinase, is involved in several distinct signaling pathways including non‐Smad pathways for TGF‐β superfamily members and inflammatory responses caused by cytokines. Conventional disruption of the murine Tab1 gene results in late gestational lethality showing intraventricular septum defects and underdeveloped lung alveoli. To gain a better understanding of the roles of TAB1 in different tissues, at different stages of development, and in pathological conditions, we generated Tab1 floxed mice in which the loxP sites flank Exons 9 and 10 to remove the C‐terminal region of TAB1 protein necessary for activation of TAK1. We demonstrate that Cre‐mediated recombination using Sox2‐Cre, a Cre line expressed in the epiblast during early embryogenesis, results in deletion of the gene and protein. These homozygous Cre‐recombined null embryos display an identical phenotype to conventional null embryos. This animal model will be useful in revealing distinct roles of TAB1 in different tissues at different stages. genesis 46:431–439, 2008. Published 2008 Wiley‐Liss, Inc.}, number={8}, journal={GENESIS}, author={Inagaki, Maiko and Komatsu, Yoshihiro and Scott, Greg and Yamada, Gen and Ray, Manas and Ninomiya-Tsuji, Jun and Mishina, Yuji}, year={2008}, month={Aug}, pages={431–439} }
 @article{prickett_ninomiya-tsuji_broglie_muratore-schroeder_shabanowitz_hunt_brautigan_2008, title={TAB4 stimulates TAK1-TAB1 phosphorylation and binds polyubiquitin to direct signaling to NF-kappa B}, volume={283}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-50349090977&partnerID=MN8TOARS}, DOI={10.1074/jbc.m800943200}, abstractNote={Responses to transforming growth factor β and multiple cytokines involve activation of transforming growth factor β-activated kinase-1 (TAK1) kinase, which activates kinases IκB kinase (IKK) and MKK3/6, leading to the parallel activation of NF-κB and p38 MAPK. Activation of TAK1 by autophosphorylation is known to involve three different TAK1-binding proteins (TABs). Here we report a protein phosphatase subunit known as type 2A phosphatase-interacting protein (TIP) that also acts as a TAB because it co-precipitates with and directly binds to TAK1, enhances TAK1 autophosphorylation at unique sites, and promotes TAK1 phosphorylation of IKKβ and signaling to NF-κB. Mass spectrometry demonstrated that co-expression of TAB4 protein significantly increased phosphorylation of four sites in TAK1, in a linker region between the kinase and TAB2/3 binding domains, and two sites in TAB1. Recombinant GST-TAB4 bound in an overlay assay directly to inactive TAK1 and activated TAK1 but not TAK1 phosphorylated in the linker sites, suggesting a bind and release mechanism. In kinase assays using TAK1 immune complexes, added GST-TAB4 selectively stimulated IKK phosphorylation. TAB4 co-precipitated polyubiquitinated proteins dependent on a Phe-Pro motif that was required to enhance phosphorylation of TAK1. TAB4 mutated at Phe-Pro dominantly interfered with IL-1β activation of NF-κB involving IKK-dependent but not p38 MAPK-dependent signaling. The results show that TAB4 binds TAK1 and polyubiquitin chains to promote specific sites of phosphorylation in TAK1-TAB1, which activates IKK signaling to NF-κB.}, number={28}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Prickett, Todd D. and Ninomiya-Tsuji, Jun and Broglie, Peter and Muratore-Schroeder, Tara L. and Shabanowitz, Jeffrey and Hunt, Donald F. and Brautigan, David L.}, year={2008}, month={Jul}, pages={19245–19254} }
 @article{omori_morioka_matsumoto_ninomiya-tsuji_2008, title={TAK1 regulates reactive oxygen species and cell death in keratinocytes, which is essential for skin integrity}, volume={283}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-54449085568&partnerID=MN8TOARS}, DOI={10.1074/jbc.M804513200}, abstractNote={Mice with a keratinocyte-specific deletion of Tak1 exhibit severe skin inflammation due to hypersensitivity to tumor necrosis factor (TNF) killing. Here we have examined the mechanisms underlying this hypersensitivity. We found that TAK1 deficiency up-regulates reactive oxygen species (ROS) resulting in cell death upon TNF or oxidative stress challenge. Because blockade of NF-κB did not increase ROS or did not sensitize cells to oxidative stress in keratinocytes TAK1 regulates ROS mainly through the mechanisms other than those mediated by NF-κB. We found that c-Jun was decreased in TAK1-deficient keratinocytes and that ectopic expression of c-Jun could partially inhibit TNF-induced increase of ROS and cell death. Finally, we show that, in an in vivo setting, the antioxidant treatment could reduce an inflammatory condition in keratinocyte-specific Tak1 deletion mice. Thus, TAK1 regulates ROS partially through c-Jun, which is important for preventing ROS-induced skin inflammation.}, number={38}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Omori, Emily and Morioka, Sho and Matsumoto, Kunihiro and Ninomiya-Tsuji, Jun}, year={2008}, month={Sep}, pages={26161–26168} }
 @article{inagaki_omori_kim_komatsu_scott_ray_yamada_matsumoto_mishina_ninomiya-tsuji_2008, title={TAK1-binding Protein 1, TAB1, Mediates Osmotic Stress-induced TAK1 Activation but Is Dispensable for TAK1-mediated Cytokine Signaling}, volume={283}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-57749122042&partnerID=MN8TOARS}, DOI={10.1074/jbc.M807574200}, abstractNote={TAK1 kinase is an indispensable intermediate in several cytokine signaling pathways including tumor necrosis factor, interleukin-1, and transforming growth factor-β signaling pathways. TAK1 also participates in stress-activated intracellular signaling pathways such as osmotic stress signaling pathway. TAK1-binding protein 1 (TAB1) is constitutively associated with TAK1 through its C-terminal region. Although TAB1 is known to augment TAK1 catalytic activity when it is overexpressed, the role of TAB1 under physiological conditions has not yet been identified. In this study, we determined the role of TAB1 in TAK1 signaling by analyzing TAB1-deficient mouse embryonic fibroblasts (MEFs). Tumor necrosis factor- and interleukin-1-induced activation of TAK1 was entirely normal in Tab1-deficient MEFs and could activate both mitogen-activated protein kinases and NF-κB. In contrast, we found that osmotic stress-induced activation of TAK1 was largely impaired in Tab1-deficient MEFs. Furthermore, we showed that the C-terminal 68 amino acids of TAB1 were sufficient to mediate osmotic stress-induced TAK1 activation. Finally, we attempted to determine the mechanism by which TAB1 activates TAK1. We found that TAK1 is spontaneously activated when the concentration is increased and that it is totally dependent on TAB1. Cell shrinkage under the osmotic stress condition increases the concentration of TAB1-TAK1 and may oligomerize and activate TAK1 in a TAB1-dependent manner. These results demonstrate that TAB1 mediates TAK1 activation only in a subset of TAK1 pathways that are mediated through spontaneous oligomerization of TAB1-TAK1.}, number={48}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Inagaki, Maiko and Omori, Emily and Kim, Jae-Young and Komatsu, Yoshihiro and Scott, Greg and Ray, Manas K. and Yamada, Gen and Matsumoto, Kunihiro and Mishina, Yuji and Ninomiya-Tsuji, Jun}, year={2008}, month={Nov}, pages={33080–33086} }
 @book{ninomiya-tsuji_2007, title={Mitochondrial Dysfunction}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-84880315825&partnerID=MN8TOARS}, DOI={10.1002/9780470285251.ch17}, abstractNote={This chapter contains sections titled: Introduction Mitochondrial Function Mitochondrial Apoptosis/Necrosis Toxicant-Induced Mitochondrial Apoptosis/Necrosis Suggested Reading}, journal={Molecular and Biochemical Toxicology, Fourth Edition}, author={Ninomiya-Tsuji, J.}, year={2007}, pages={319–332} }
 @article{huangfu_matsumoto_ninomiya-tsuji_2007, title={Osmotic stress blocks NF-kappa B-dependent in inflammatory responses by inhibiting ubiquitination of I kappa B}, volume={581}, ISSN={["1873-3468"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-36549023962&partnerID=MN8TOARS}, DOI={10.1016/j.febslet.2007.11.002}, abstractNote={The inhibitory effects of hypertonic conditions on immune responses have been described in clinical studies; however, the molecular mechanism underlying this phenomenon has yet to be defined. Here we investigate osmotic stress‐mediated modification of the NF‐κB pathway, a central signaling pathway in inflammation. We unexpectedly found that osmotic stress could activate IκBα kinase but did not activate NF‐κB. Osmotic stress‐induced phosphorylated IκBα was not ubiquitinated, and osmotic stress inhibited interleukin 1‐induced ubiquitination of IκBα and ultimately blocked expression of cytokine/chemokines. Thus, blockage of IκBα ubiquitination is likely to be a major mechanism for inhibition of inflammation by hypertonic conditions.}, number={29}, journal={FEBS LETTERS}, author={HuangFu, Wei-Chun and Matsumoto, Kunihiro and Ninomiya-Tsuji, Jun}, year={2007}, month={Dec}, pages={5549–5554} }
 @article{kajino_omori_ishii_matsumoto_ninomiya-tsuji_2007, title={TAK1 MAPK kinase kinase mediates transforming growth factor-beta signaling by targeting SnoN oncoprotein for degradation}, volume={282}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-34248147701&partnerID=MN8TOARS}, DOI={10.1074/jbc.M700875200}, abstractNote={Abstract Transforming growth factor-β (TGF-β) regulates a variety of physiologic processes through essential intracellular mediators Smads. The SnoN oncoprotein is an inhibitor of TGF-β signaling. SnoN recruits transcriptional repressor complex to block Smad-dependent transcriptional activation of TGF-β-responsive genes. Following TGF-β stimulation, SnoN is rapidly degraded, thereby allowing the activation of TGF-β target genes. Here, we report the role of TAK1 as a SnoN protein kinase. TAK1 interacted with and phosphorylated SnoN, and this phosphorylation regulated the stability of SnoN. Inactivation of TAK1 prevented TGF-β-induced SnoN degradation and impaired induction of the TGF-β-responsive genes. These data suggest that TAK1 modulates TGF-β-dependent cellular responses by targeting SnoN for degradation.}, number={13}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Kajino, Taisuke and Omori, Emily and Ishii, Shunsuke and Matsumoto, Kunihiro and Ninomiya-Tsuji, Jun}, year={2007}, month={Mar}, pages={9475–9481} }
 @article{kim_omori_matsumoto_nunez_ninomiya-tsuji_2008, title={TAK1 is a central mediator of NOD2 signaling in epidermal cells}, volume={283}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-38049153165&partnerID=MN8TOARS}, DOI={10.1074/jbc.M704746200}, abstractNote={Muramyl dipeptide (MDP) is a peptidoglycan moiety derived from commensal and pathogenic bacteria, and a ligand of its intracellular sensor NOD2. Mutations in NOD2 are highly associated with Crohn disease, which is characterized by dysregulated inflammation in the intestine. However, the mechanism linking abnormality of NOD2 signaling and inflammation has yet to be elucidated. Here we show that transforming growth factor β-activated kinase 1 (TAK1) is an essential intermediate of NOD2 signaling. We found that TAK1 deletion completely abolished MDP-NOD2 signaling, activation of NF-κB and MAPKs, and subsequent induction of cytokines/chemokines in keratinocytes. NOD2 and its downstream effector RICK associated with and activated TAK1. TAK1 deficiency also abolished MDP-induced NOD2 expression. Because mice with epidermis-specific deletion of TAK1 develop severe inflammatory conditions, we propose that TAK1 and NOD2 signaling are important for maintaining normal homeostasis of the skin, and its ablation may impair the skin barrier function leading to inflammation.}, number={1}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Kim, Jae-Young and Omori, Emily and Matsumoto, Kunihiro and Nunez, Gabriel and Ninomiya-Tsuji, Jun}, year={2008}, month={Jan}, pages={137–144} }
 @article{huangfu_omori_akira_matsumoto_ninomiya-tsuji_2006, title={Osmotic Stress Activates the TAK1-JNK Pathway While Blocking TAK1-mediated NF-κB Activation}, volume={281}, ISSN={0021-9258 1083-351X}, url={http://dx.doi.org/10.1074/JBC.M603627200}, DOI={10.1074/JBC.M603627200}, abstractNote={Osmotic stress activates MAPKs, including JNK and p38, which play important roles in cellular stress responses. Transforming growth factor-β-activated kinase 1 (TAK1) is a member of the MAPK kinase kinase (MAPKKK) family and can activate JNK and p38. TAK1 can also activate IκB kinase (IKK) that leads to degradation of IκB and subsequent NF-κB activation. We found that TAK1 is essential for osmotic stress-induced activation of JNK but is not an exclusive mediator of p38 activation. Furthermore, we found that although TAK1 was highly activated upon osmotic stress, it could not induce degradation of IκB or activation of NF-κB. These results suggest that TAK1 activity is somehow modulated to function specifically in osmotic stress signaling, leading to the activation of JNK but not of IKK. To elucidate the mechanism underlying this modulation, we screened for potential TAK1-binding proteins. We found that TAO2 (thousand-and-one amino acid kinase 2) associates with TAK1 and can inhibit TAK1-mediated activation of NF-κB but not of JNK. We observed that TAO2 can interfere with the interaction between TAK1 and IKK and thus may regulate TAK1 function. TAK1 is activated by many distinct stimuli, including cytokines and stresses, and regulation by TAO2 may be important to activate specific intracellular signaling pathways that are unique to osmotic stress.}, number={39}, journal={Journal of Biological Chemistry}, publisher={American Society for Biochemistry & Molecular Biology (ASBMB)}, author={HuangFu, Wei-Chun and Omori, Emily and Akira, Shizuo and Matsumoto, Kunihiro and Ninomiya-Tsuji, Jun}, year={2006}, month={Aug}, pages={28802–28810} }
 @article{huangfu_omori_akira_matsumoto_ninomiya-tsuji_2006, title={Osmotic stress activates the TAK1-JNK pathway while blocking TAK1-mediated NF-kappa B activation - TAO2 regulates TAK1 pathways}, volume={281}, DOI={10.1014/jbc.M60362/200}, number={39}, journal={Journal of Biological Chemistry}, author={Huangfu, W. C. and Omori, E. and Akira, S. and Matsumoto, K. and Ninomiya-Tsuji, Jun}, year={2006}, pages={28802–28810} }
 @article{kajino_ren_iemura_natsume_stefansson_brautigan_matsumoto_ninomiya-tsuji_2006, title={Protein phosphatase 6 down-regulates TAK1 kinase activation in the IL-1 signaling pathway}, volume={281}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33845994781&partnerID=MN8TOARS}, DOI={10.1074/jbc.M608155200}, abstractNote={TAK1 (transforming growth factor β-activated kinase 1) is a serine/threonine kinase that is a mitogen-activated protein kinase kinase kinase and an essential intracellular signaling component in inflammatory signaling pathways. Upon stimulation of cells with inflammatory cytokines, TAK1 binds proteins that stimulate autophosphorylation within its activation loop and is thereby catalytically activated. This activation is transient; it peaks within a couple of minutes and is subsequently down-regulated rapidly to basal levels. The mechanism of down-regulation of TAK1 has not yet been elucidated. In this study, we found that toxin inhibition of type 2A protein phosphatases greatly enhances interleukin 1 (IL-1)-dependent phosphorylation of Thr-187 in the TAK1 activation loop as well as the catalytic activity of TAK1. From proteomic analysis of TAK1-binding proteins, we identified protein phosphatase 6 (PP6), a type-2A phosphatase, and demonstrated that PP6 associated with and inactivated TAK1 by dephosphorylation of Thr-187. Ectopic and endogenous PP6 co-precipitated with TAK1, and expression of PP6 reduced IL-1 activation of TAK1 but did not affect osmotic activation of MLK3, another MAPKKK. Reduction of PP6 expression by small interfering RNA enhances IL-1-induced phosphorylation of Thr-187 in TAK1. Enhancement occurred without change in levels of PP2A showing specificity for PP6. Our results demonstrate that PP6 specifically down-regulates TAK1 through dephosphorylation of Thr-187 in the activation loop, which is likely important for suppressing inflammatory responses via TAK1 signaling pathways.}, number={52}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Kajino, Taisuke and Ren, Hong and Iemura, Shun-ichiro and Natsume, Tohru and Stefansson, Bjarki and Brautigan, David L. and Matsumoto, Kunihiro and Ninomiya-Tsuji, Jun}, year={2006}, month={Dec}, pages={39891–39896} }
 @article{uemura_kajino_sanjo_sato_akira_matsumoto_ninomiya-tsuji_2006, title={TAK1 is a component of the Epstein-Barr virus LMP1 complex and is essential for activation of JNK but not of NF-kappa B}, volume={281}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33646359441&partnerID=MN8TOARS}, DOI={10.1074/jbc.M509834200}, abstractNote={Epstein-Barr virus latent membrane protein 1 (LMP1) activates NF-κB and c-Jun N-terminal kinase (JNK), which is essential for LMP1 oncogenic activity. Genetic analysis has revealed that tumor necrosis factor receptor-associated factor 6 (TRAF6) is an indispensable intermediate of LMP1 signaling leading to activation of both NF-κB and JNK. However, the mechanism by which LMP1 engages TRAF6 for activation of NF-κB and JNK is not well understood. Here we demonstrate that TAK1 mitogen-activated protein kinase kinase kinase and TAK1-binding protein 2 (TAB2), together with TRAF6, are recruited to LMP1 through its N-terminal transmembrane region. The C-terminal cytoplasmic region of LMP1 facilitates the assembly of this complex and enhances activation of JNK. In contrast, IκB kinase γ is recruited through the C-terminal cytoplasmic region and this is essential for activation of NF-κB. Furthermore, we found that ablation of TAK1 resulted in the loss of LMP1-induced activation of JNK but not of NF-κB. These results suggest that an LMP1-associated complex containing TRAF6, TAB2, and TAK1 plays an essential role in the activation of JNK. However, TAK1 is not an exclusive intermediate for NF-κB activation in LMP1 signaling.}, number={12}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Uemura, N and Kajino, T and Sanjo, H and Sato, S and Akira, S and Matsumoto, K and Ninomiya-Tsuji, J}, year={2006}, month={Mar}, pages={7863–7872} }
 @article{omori_matsumoto_sanjo_sato_akira_smart_ninomiya-tsuji_2006, title={TAK1 is a master regulator of epidermal homeostasis involving skin inflammation and apoptosis}, volume={281}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33745851830&partnerID=MN8TOARS}, DOI={10.1074/jbc.M603384200}, abstractNote={Transforming growth factor β-activated kinase 1 (TAK1) functions downstream of inflammatory cytokines to activate c-Jun N-terminal kinase (JNK) as well as NF-κB in several cell types. However, the functional role of TAK1 in an in vivo setting has not been determined. Here we have demonstrated that TAK1 is the major regulator of skin inflammation as well as keratinocyte death in vivo. Epidermal-specific deletion of TAK1 causes a severe inflammatory skin condition by postnatal day 6-8. The mutant skin also exhibits massive keratinocyte death. Analysis of keratinocytes isolated from the mutant skin revealed that TAK1 deficiency results in a striking increase in apoptosis in response to tumor necrosis factor (TNF). TAK1-deficient keratinocytes cannot activate NF-κB or JNK upon TNF treatment. These results suggest that TNF induces TAK1-deficient keratinocyte death because of the lack of NF-κB (and possibly JNK)-mediated cell survival signaling. Finally, we have shown that deletion of the TNF receptor can largely rescue keratinocyte death as well as inflammatory skin condition in epidermal-specific TAK1-deficient mice. Our results demonstrate that TAK1 is a master regulator of TNF signaling in skin and regulates skin inflammation and keratinocyte death.}, number={28}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Omori, Emily and Matsumoto, Kunihiro and Sanjo, Hideki and Sato, Shintaro and Akira, Shizuo and Smart, Robert C. and Ninomiya-Tsuji, Jun}, year={2006}, month={Jul}, pages={19610–19617} }
 @article{sato_sanjo_tsujimura_ninomiya-tsuji_yamamoto_kawai_takeuchi_akira_2006, title={TAK1 is indispensable for development of T cells and prevention of colitis by the generation of regulatory T cells}, volume={18}, ISSN={["1460-2377"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-33748999526&partnerID=MN8TOARS}, DOI={10.1093/intimm/dxl082}, abstractNote={Transforming growth factor (TGF)-beta-activating kinase 1 (TAK1) is critical for Toll-like receptor- and tumor necrosis factor-mediated cellular responses. In B cells, TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs), but not nuclear factor-kappaB (NF-kappaB), in antigen receptor signaling. In this study, we generate T cell-specific TAK1-deficient (Lck(Cre/(+))Tak1(flox/flox)) mice and show that TAK1 is indispensable for the maintenance of peripheral CD4 and CD8 T cells. In thymocytes, TAK1 is essential for TCR-mediated activation of both NF-kappaB and MAPKs. Additionally, Lck(Cre/(+))Tak1(flox/flox) mice developed colitis as they aged. In these mice, accumulations of activated/memory T cells as well as B cells were observed. Development of regulatory T (Treg) cells in thymus was abrogated in Lck(Cre/(+))Tak1(flox/flox) mice, suggesting that the loss of Treg cells is the cause of the disease. Together, the results show that TAK1, by controlling the generation of central Treg cells, is important for preventing spontaneously developing colitis.}, number={10}, journal={INTERNATIONAL IMMUNOLOGY}, author={Sato, Shintaro and Sanjo, Hideki and Tsujimura, Tohru and Ninomiya-Tsuji, Jun and Yamamoto, Masahiro and Kawai, Taro and Takeuchi, Osamu and Akira, Shizuo}, year={2006}, month={Oct}, pages={1405–1411} }
 @article{ninomiya-tsuji_matsumoto_2006, title={Tab1}, volume={3}, ISSN={1477-5921}, url={http://dx.doi.org/10.1038/mp.a002247.01}, DOI={10.1038/mp.a002247.01}, journal={AfCS-Nature Molecule Pages}, publisher={Springer Science and Business Media LLC}, author={Ninomiya-Tsuji, Jun and Matsumoto, Kunihiro}, year={2006}, month={Mar} }
 @article{ninomiya-tsuji_matsumoto_2006, title={Tak1}, volume={1}, ISSN={1477-5921}, url={http://dx.doi.org/10.1038/mp.a002249.01}, DOI={10.1038/mp.a002249.01}, journal={AfCS-Nature Molecule Pages}, publisher={Springer Science and Business Media LLC}, author={Ninomiya-Tsuji, Jun and Matsumoto, Kunihiro}, year={2006}, month={Jan} }
 @article{li_miller_ninomiya-tsuji_russell_young_2005, title={AMP-activated protein kinase activates p38 mitogen-activated protein kinase by increasing recruitment of p38 MAPK to TAB1 in the ischemic heart}, volume={97}, ISSN={["1524-4571"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-27644477357&partnerID=MN8TOARS}, DOI={10.1161/01.RES.0000187458.77026.10}, abstractNote={
            AMP-activated protein kinase (AMPK) promotes glucose transport, maintains ATP stores, and prevents injury and apoptosis during ischemia. AMPK has several direct molecular targets in the heart but also may interact with other stress-signaling pathways. This study examined the role of AMPK in the activation of the p38 mitogen-activated protein kinase (MAPK). In isolated heart muscles, the AMPK activator 5-aminoimidazole-4-carboxy-amide-1-β-
            d
            -ribofuranoside (AICAR) increased p38 MAPK activation. In AMPK-deficient mouse hearts, expressing a kinase-dead (KD) α
            2
            catalytic subunit, p38 MAPK activation was markedly reduced during low-flow ischemia (2.3- versus 7-fold in wild-type hearts,
            P
            <0.01) and was similarly reduced during severe no-flow ischemia in KD hearts (
            P
            <0.01 versus ischemic wild type). Knockout of the p38 MAPK upstream kinase, MAPK kinase 3 (MKK3), did not affect ischemic activation of either AMPK or p38 MAPK in transgenic mkk3
            −/−
            mouse hearts. Ischemia increased p38 MAPK recruitment to transforming growth factor-β-activated protein kinase 1–binding protein 1 (TAB1), a scaffold protein that promotes p38 MAPK autophosphorylation. Moreover, TAB1 was associated with the α
            2
            catalytic subunit of AMPK. p38 MAPK recruitment to TAB1/AMPK complexes required AMPK activation and was reduced in ischemic AMPK-deficient transgenic mouse hearts. The potential role of p38 MAPK in mediating the downstream action of AMPK to promote glucose transport was also assessed. The p38 MAPK inhibitor SB203580 partially inhibited both AICAR- and hypoxia-stimulated glucose uptake and GLUT4 translocation. Activation of p38 MAPK by anisomycin also increased glucose transport in heart muscles. Thus, AMPK has an important role in promoting p38 MAPK activation in the ischemic heart by inducing p38 MAPK autophosphorylation through interaction with the scaffold protein TAB1.
          }, number={9}, journal={CIRCULATION RESEARCH}, author={Li, J and Miller, EJ and Ninomiya-Tsuji, J and Russell, RR and Young, LH}, year={2005}, month={Oct}, pages={872–879} }
 @article{sato_sanjo_takeda_ninomiya-tsuji_yamamoto_kawai_matsumoto_takeuchi_akira_2005, title={Essential function for the kinase TAK1 in innate and adaptive immune responses}, volume={6}, ISSN={["1529-2916"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-27544434183&partnerID=MN8TOARS}, DOI={10.1038/ni1255}, abstractNote={Transforming growth factor-beta-activated kinase 1 (TAK1) has been linked to interleukin 1 receptor and tumor necrosis factor receptor signaling. Here we generated mouse strains with conditional expression of a Map3k7 allele encoding part of TAK1. TAK1-deficient embryonic fibroblasts demonstrated loss of responses to interleukin 1beta and tumor necrosis factor. Studies of mice with B cell-specific TAK1 deficiency showed that TAK1 was indispensable for cellular responses to Toll-like receptor ligands, CD40 and B cell receptor crosslinking. In addition, antigen-induced immune responses were considerably impaired in mice with B cell-specific TAK1 deficiency. TAK1-deficient cells failed to activate transcription factor NF-kappaB and mitogen-activated protein kinases in response to interleukin 1beta, tumor necrosis factor and Toll-like receptor ligands. However, TAK1-deficient B cells were able to activate NF-kappaB but not the kinase Jnk in response to B cell receptor stimulation. These results collectively indicate that TAK1 is key in the cellular response to a variety of stimuli.}, number={11}, journal={NATURE IMMUNOLOGY}, author={Sato, S and Sanjo, H and Takeda, K and Ninomiya-Tsuji, J and Yamamoto, M and Kawai, T and Matsumoto, K and Takeuchi, O and Akira, S}, year={2005}, month={Nov}, pages={1087–1095} }
 @article{kishida_sanjo_akira_matsumoto_ninomiya-tsuji_2005, title={TAK1-binding protein 2 facilitates ubiquitination of TRAF6 and assembly of TRAF6 with IKK in the IL-1 signaling pathway}, volume={10}, ISSN={["1365-2443"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-17844393079&partnerID=MN8TOARS}, DOI={10.1111/j.1365-2443.2005.00852.x}, abstractNote={TAK1 mitogen‐activated protein kinase kinase kinase participates in the Interleukin‐1 (IL‐1) signaling pathway by mediating activation of JNK, p38, and NF‐κB. TAK1‐binding protein 2 (TAB2) was previously identified as an adaptor that links TAK1 to an upstream signaling intermediate, tumor necrosis factor receptor‐associated factor 6 (TRAF6). Recently, ubiquitination of TRAF6 was shown to play an essential role in the activation of TAK1. However, the mechanism by which IL‐1 induces TRAF6 ubiquitination remains to be elucidated. Here we report that TAB2 functions to facilitate TRAF6 ubiquitination and thereby mediates IL‐1‐induced cellular events. A conserved ubiquitin binding domain in TAB2, the CUE domain, is important for this function. We also found that TAB2 promotes the assembly of TRAF6 with a downstream kinase, IκB kinase (IKK). These results show that TAB2 acts as a multifunctional signaling molecule, facilitating both IL‐1‐dependent TRAF6 ubiquitination and assembly of the IL‐1 signaling complex.}, number={5}, journal={GENES TO CELLS}, author={Kishida, S and Sanjo, H and Akira, S and Matsumoto, K and Ninomiya-Tsuji, J}, year={2005}, month={May}, pages={447–454} }
 @article{safwat_ninomiya-tsuji_gore_miller_2005, title={Transforming growth factor beta-activated kinase 1 is a key mediator of ovine follicle-stimulating hormone beta-subunit expression}, volume={146}, ISSN={["1945-7170"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-26844553319&partnerID=MN8TOARS}, DOI={10.1210/en.2005-0457}, abstractNote={FSH, a key regulator of gonadal function, contains a β-subunit (FSHβ) that is transcriptionally induced by activin, a member of the TGFβ-superfamily. This study used 4.7 kb of the ovine FSHβ-promoter linked to luciferase (oFSHβLuc) plus a well-characterized activin-responsive construct, p3TPLuc, to investigate the hypothesis that Smad3, TGFβ-activated kinase 1 (TAK1), or both cause activin-mediated induction of FSH. Overexpression of either Smad3 or TAK1 induced oFSHβLuc in gonadotrope-derived LβT2 cells as much as activin itself. Induction of p3TPLuc by activin is known to require Smad3 activation in many cell types, and this was true in LβT2 cells, where 10-fold induction by activin (2–8 h after activin treatment) was blocked more than 90% by two dominant negative (DN) inhibitors of Smad3 [DN-Smad3 (3SA) and DN-Smad3 (D407E)]. By contrast, 6.5-fold induction of oFSHβLuc by activin (10–24 h after activin treatment) was not blocked by either DN-Smad inhibitor, suggesting that activation of Smad3 did not trigger induction of oFSHβLuc. By contrast, inhibition of TAK1 by a DN-TAK1 construct led to a 50% decrease in activin-mediated induction of oFSHβLuc, and a specific inhibitor of TAK1 (5Z-7-Oxozeanol) blocked induction by 100%, indicating that TAK1 is necessary for activin induction of oFSHβLuc. Finally, inhibiting p38-MAPK (often activated by TAK1) blocked induction of oFSHβLuc by 60%. In conclusion, the data presented here indicate that activation of TAK1 (and probably p38-MAPK), but not Smad3, is necessary for triggering induction of oFSHβ by activin.}, number={11}, journal={ENDOCRINOLOGY}, author={Safwat, N and Ninomiya-Tsuji, J and Gore, AJ and Miller, WL}, year={2005}, month={Nov}, pages={4814–4824} }
 @article{akiyama_yonezawa_kudo_li_wang_ito_yoshioka_ninomiya-tsuji_matsumoto_kanamaru_et al._2004, title={Activation mechanism of c-Jun amino-terminal kinase in the course of neural differentiation of P19 embryonic carcinoma cells}, volume={279}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-4344606739&partnerID=MN8TOARS}, DOI={10.1074/jbc.M406610200}, abstractNote={P19 embryonic carcinoma cells, a model system for studying early development and differentiation, can differentiate into neurons and primitive endoderm-like cells depending on the culture conditions. We have previously reported that the activation of c-Jun amino-terminal kinase (JNK) is required for the retinoic acid-induced neural differentiation of P19 cells. However, the signaling pathway(s) responsible for the activation of JNK has not been known. In this study, we demonstrated that activities of MAPK kinase 4 (MKK4) and TAK1, one of the upstream kinases of MKK4, were enhanced in the neurally differentiating cells. Inhibition of the neural differentiation by an overexpression of protein phosphatase 2Cϵ, an inactivator of TAK1, suggested a critical role of the TAK1 signaling pathway during the differentiation. Confocal microscopic analysis indicated that TAK1, phospho-MKK4, and phospho-JNK were colocalized with tubulin in the neurites and localized also in the nuclei of the differentiating cells. In contrast, two TAK1-binding proteins, TAB1 and TAB2, which are involved in the activation of TAK1, were localized in the neurites and the nuclei of the differentiating cells, respectively. These results suggest that two distinct TAK1-MKK4-JNK signaling pathways are independently activated at the different intracellular locations and may participate in the regulation of the neural differentiation of P19 cells.}, number={35}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Akiyama, S and Yonezawa, T and Kudo, TA and Li, MG and Wang, H and Ito, M and Yoshioka, K and Ninomiya-Tsuji, J and Matsumoto, K and Kanamaru, R and et al.}, year={2004}, month={Aug}, pages={36616–36620} }
 @article{takeda_matsuzawa_nishitoh_tobiume_kishida_ninomiya‐tsuji_matsumoto_ichijo_2004, title={Involvement of ASK1 in Ca
            2+
            ‐induced p38 MAP kinase activation}, volume={5}, ISSN={1469-221X 1469-3178}, url={http://dx.doi.org/10.1038/sj.embor.7400072}, DOI={10.1038/sj.embor.7400072}, abstractNote={The mammalian mitogen‐activated protein (MAP) kinase kinase kinase apoptosis signal‐regulating kinase 1 (ASK1) is a pivotal component in cytokine‐ and stress‐induced apoptosis. It also regulates cell differentiation and survival through p38 MAP kinase activation. Here we show that Ca2+ signalling regulates the ASK1–p38 MAP kinase cascade. Ca2+ influx evoked by membrane depolarization in primary neurons and synaptosomes induced activation of p38, which was impaired in those derived from ASK1‐deficient mice. Ca2+/calmodulin‐dependent protein kinase type II (CaMKII) activated ASK1 by phosphorylation. Moreover, p38 activation induced by the expression of constitutively active CaMKII required endogenous ASK1. Thus, ASK1 is a critical intermediate of Ca2+ signalling between CaMKII and p38 MAP kinase.}, number={2}, journal={EMBO reports}, publisher={EMBO}, author={Takeda, Kohsuke and Matsuzawa, Atsushi and Nishitoh, Hideki and Tobiume, Kei and Kishida, Satoshi and Ninomiya‐Tsuji, Jun and Matsumoto, Kunihiro and Ichijo, Hidenori}, year={2004}, month={Jan}, pages={161–166} }
 @article{kanei-ishii_ninomiya-tsuji_tanikawa_nomura_ishitani_kishida_kokura_kurahashi_ichikawa-iwata_kim_et al._2004, title={Wnt-1 signal induces, phosphorylation and degradation of c-Myb protein via TAK1, HIPK2, and NLK}, volume={18}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-11144355861&partnerID=MN8TOARS}, DOI={10.1101/gad.1170604}, abstractNote={The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes. However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear. Here we report that c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-β-activated kinase), HIPK2 (homeodomain-interacting protein kinase 2), and NLK (Nemo-like kinase). Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK2 and the mitogen-activated protein (MAP) kinase-like kinase NLK. NLK binds directly to c-Myb together with HIPK2, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells. The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps.}, number={7}, journal={Genes and Development}, author={Kanei-Ishii, C. and Ninomiya-Tsuji, J. and Tanikawa, J. and Nomura, T. and Ishitani, T. and Kishida, S. and Kokura, K. and Kurahashi, T. and Ichikawa-Iwata, E. and Kim, Y. and et al.}, year={2004}, pages={816–829} }
 @article{ono_ohtomo_ninomiya-tsuji_tsuchiya_2003, title={A dominant negative TAK1 inhibits cellular fibrotic responses induced by TGF-beta}, volume={307}, ISSN={["0006-291X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0038351964&partnerID=MN8TOARS}, DOI={10.1016/S0006-291X(03)01207-5}, abstractNote={Transforming growth factor-beta (TGF-beta) is crucially virulent in the progression of fibrotic disorders. TAK1 (TGF-beta activated kinase 1) is one of the mitogen-activated kinase kinase kinase (MAPKKK) that is involved in TGF-beta signal transduction. To elucidate the importance of TAK1 in TGF-beta-induced fibrotic marker expression, we investigated whether dominant negative TAK1 could suppress TGF-beta signaling. Based on the finding that TAB1 (TAK1 binding protein 1) binding to TAK1 is required for TAK1 activation, a minimal portion of TAK1 lacking kinase activity that binds to TAB1 was designed as a TAK1 dominant negative inhibitor (TAK1-DN). The effect of TAK1-DN was assessed in the cells that respond to TGF-beta stimulation and that lead to the increase in production of extracellular matrix (ECM) proteins. TAK1-DN, indeed, decreased the ECM protein production, indicating that TAK1-DN retains the ability to intercept the TGF-beta signaling effectively.}, number={2}, journal={BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS}, author={Ono, K and Ohtomo, T and Ninomiya-Tsuji, J and Tsuchiya, M}, year={2003}, month={Jul}, pages={332–337} }
 @article{ninomiya-tsuji_kajino_ono_ohtomo_matsumoto_shiina_mihara_tsuchiya_matsumoto_2003, title={A resorcylic acid lactone, 5Z-7-oxozeaenol, prevents inflammation by inhibiting the catalytic activity of TAK1 MAPK kinase kinase}, volume={278}, ISSN={["0021-9258"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0037903145&partnerID=MN8TOARS}, DOI={10.1074/jbc.M207453200}, abstractNote={TAK1, a member of the mitogen-activated kinase kinase kinase (MAPKKK) family, participates in proinflammatory cellular signaling pathways by activating JNK/p38 MAPKs and NF-κB. To identify drugs that prevent inflammation, we screened inhibitors of TAK1 catalytic activity. We identified a natural resorcylic lactone of fungal origin, 5Z-7-oxozeaenol, as a highly potent inhibitor of TAK1. This compound did not effectively inhibit the catalytic activities of the MEKK1 or ASK1 MAPKKKs, suggesting that 5Z-7-oxozeaenol is a selective inhibitor of TAK1. In cell culture, 5Z-7-oxozeaenol blocked interleukin-1-induced activation of TAK1, JNK/p38 MAPK, IκB kinases, and NF-κB, resulting in inhibition of cyclooxgenase-2 production. Furthermore, in vivo 5Z-7-oxozeaenol was able to inhibit picryl chloride-induced ear swelling. Thus, 5Z-7-oxozeaenol blocks proinflammatory signaling by selectively inhibiting TAK1 MAPKKK.}, number={20}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Ninomiya-Tsuji, J and Kajino, T and Ono, K and Ohtomo, T and Matsumoto, M and Shiina, M and Mihara, M and Tsuchiya, M and Matsumoto, K}, year={2003}, month={May}, pages={18485–18490} }
 @article{ishitani_ninomiya-tsuji_matsumoto_2003, title={Regulation of lymphoid enhancer factor 1/T-cell factor by mitogen-activated protein kinase-related Nemo-like kinase-dependent phosphorylation in Wnt/β-catenin signaling}, volume={23}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0037313260&partnerID=MN8TOARS}, DOI={10.1128/MCB.23.4.1379-1389.2003}, abstractNote={The Wnt/beta-catenin signaling pathway regulates many developmental processes by modulating gene expression. Wnt signaling induces the stabilization of cytosolic beta-catenin, which then associates with lymphoid enhancer factor and T-cell factor (LEF-1/TCF) to form a transcription complex that activates Wnt target genes. Previously, we have shown that a specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase kinase kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) suppresses Wnt signaling. In this study, we investigated the relationships among NLK, beta-catenin, and LEF-1/TCF. We found that NLK interacts directly with LEF-1/TCF and indirectly with beta-catenin via LEF-1/TCF to form a complex. NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region. Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK. Phosphorylation of TCF-4 by NLK inhibited DNA binding by the beta-catenin-TCF-4 complex. However, this inhibition was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines. These results suggest that NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity.}, number={4}, journal={Molecular and Cellular Biology}, author={Ishitani, T. and Ninomiya-Tsuji, J. and Matsumoto, K.}, year={2003}, pages={1379–1389} }
 @article{li_katsura_nomiyama_komaki_ninomiya-tsuji_matsumoto_kobayashi_tamura_2003, title={Regulation of the interleukin-1-induced signaling pathways by a novel member of the protein phosphatase 2C family (PP2C epsilon)}, volume={278}, ISSN={["1083-351X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0037809234&partnerID=MN8TOARS}, DOI={10.1074/jbc.M211474200}, abstractNote={Although TAK1 signaling plays essential roles in eliciting cellular responses to interleukin-1 (IL-1), a proinflammatory cytokine, how the IL-1-TAK1 signaling pathway is positively and negatively regulated remains poorly understood. In this study, we investigated the possible role of a novel protein phosphatase 2C (PP2C) family member, PP2Cε, in the regulation of the IL-1-TAK1 signaling pathway. PP2Cε was composed of 303 amino acids, and the overall similarity of amino acid sequence between PP2Cε and PP2Cα was found to be 26%. Ectopic expression of PP2Cε inhibited the IL-1- and TAK1-induced activation of mitogen-activated protein kinase kinase 4 (MKK4)-c-Jun N-terminal kinase or MKK3-p38 signaling pathway. PP2Cε dephosphorylated TAK1 in vitro. Co-immunoprecipitation experiments indicated that PP2Cε associates stably with TAK1 and attenuates the binding of TAK1 to MKK4 or MKK6. Ectopic expression of a phosphatase-negative mutant of PP2Cε, PP2Cε(D/A), which acted as a dominant negative form, enhanced both the association between TAK1 and MKK4 or MKK6 and the TAK1-induced activation of an AP-1 reporter gene. The association between PP2Cε and TAK1 was transiently suppressed by IL-1 treatment of the cells. Taken together, these results suggest that, in the absence of IL-1-induced signal, PP2Cε contributes to keeping the TAK1 signaling pathway in an inactive state by associating with and dephosphorylating TAK1.}, number={14}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Li, MG and Katsura, K and Nomiyama, H and Komaki, K and Ninomiya-Tsuji, J and Matsumoto, K and Kobayashi, T and Tamura, S}, year={2003}, month={Apr}, pages={12013–12021} }
 @article{ishitani_takaesu_ninomiya-tsuji_shibuya_gaynor_matsumoto_2003, title={Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling}, volume={22}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0346243941&partnerID=MN8TOARS}, DOI={10.1093/emboj/cdg605}, number={23}, journal={EMBO Journal}, author={Ishitani, T. and Takaesu, G. and Ninomiya-Tsuji, J. and Shibuya, H. and Gaynor, R.B. and Matsumoto, K.}, year={2003}, pages={6277–6288} }
 @article{sanjo_takeda_tsujimura_ninomiya-tsuji_matsumoto_akira_2003, title={TAB2 is essential for prevention of apoptosis in fetal liver but not for interleukin-1 signaling}, volume={23}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0037313041&partnerID=MN8TOARS}, DOI={10.1128/MCB.23.4.1231-1238.2003}, abstractNote={The proinflammatory cytokine interleukin-1 (IL-1) transmits a signal via several critical cytoplasmic proteins such as MyD88, IRAKs and TRAF6. Recently, serine/threonine kinase TAK1 and TAK1 binding protein 1 and 2 (TAB1/2) have been identified as molecules involved in IL-1-induced TRAF6-mediated activation of AP-1 and NF-kappa B via mitogen-activated protein (MAP) kinases and I kappa B kinases, respectively. However, their physiological functions remain to be clarified. To elucidate their roles in vivo, we generated TAB2-deficient mice. The TAB2 deficiency was embryonic lethal due to liver degeneration and apoptosis. This phenotype was similar to that of NF-kappa B p65-, IKK beta-, and NEMO/IKK gamma-deficient mice. However, the IL-1-induced activation of NF-kappa B and MAP kinases was not impaired in TAB2-deficient embryonic fibroblasts. These findings demonstrate that TAB2 is essential for embryonic development through prevention of liver apoptosis but not for the IL-1 receptor-mediated signaling pathway.}, number={4}, journal={Molecular and Cellular Biology}, author={Sanjo, H. and Takeda, K. and Tsujimura, T. and Ninomiya-Tsuji, J. and Matsumoto, K. and Akira, S.}, year={2003}, pages={1231–1238} }
 @article{takaesu_surabhi_park_ninomiya-tsuji_matsumoto_gaynor_2003, title={TAK1 is critical for I kappa B kinase-mediated activation of the NF-kappa B pathway}, volume={326}, ISSN={["0022-2836"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0037422858&partnerID=MN8TOARS}, DOI={10.1016/S0022-2836(02)01404-3}, abstractNote={Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.}, number={1}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Takaesu, G and Surabhi, RM and Park, KJ and Ninomiya-Tsuji, J and Matsumoto, K and Gaynor, RB}, year={2003}, month={Feb}, pages={105–115} }
 @article{jiang_ninomiya-tsuji_qian_matsumoto_li_2002, title={Interleukin-1 (IL-1) receptor-associated kinase-dependent IL-1-induced signaling complexes phosphorylate TAK1 and TAB2 at the plasma membrane and activate TAK1 in the cytosol}, volume={22}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0036788753&partnerID=MN8TOARS}, DOI={10.1128/MCB.22.20.7158-7167.2002}, abstractNote={Interleukin-1 (IL-1) receptor-associated kinase (IRAK) plays an important role in the sequential formation and activation of IL-1-induced signaling complexes.Previous studies showed that IRAK is recruited to the IL-1-receptor complex, where it is hyperphosphorylated.We now find that the phosphorylated IRAK in turn recruits TRAF6 to the receptor complex (complex I), which differs from the previous concept that IRAK interacts with TRAF6 after it leaves the receptor.IRAK then brings TRAF6 to TAK1, TAB1, and TAB2, which are preassociated on the membrane before stimulation to form the membrane-associated complex II.The formation of complex II leads to the phosphorylation of TAK1 and TAB2 on the membrane by an unknown kinase, followed by the dissociation of TRAF6-TAK1-TAB1-TAB2 (complex III) from IRAK and consequent translocation of complex III to the cytosol.The formation of complex III and its interaction with additional cytosolic factors lead to the activation of TAK1, resulting in NF-B and JNK activation.Phosphorylated IRAK remains on the membrane and eventually is ubiquitinated and degraded.Taken together, the new data reveal that IRAK plays a critical role in mediating the association and dissociation of IL-1-induced signaling complexes, functioning as an organizer and transporter in IL-1-dependent signaling.}, number={20}, journal={Molecular and Cellular Biology}, author={Jiang, Z. and Ninomiya-Tsuji, J. and Qian, Y. and Matsumoto, K. and Li, X.}, year={2002}, pages={7158–7167} }
 @article{mizukami_takaesu_akatsuka_sakurai_ninomiya-tsuji_matsumoto_sakurai_2002, title={Receptor activator of NF-κB ligand (RANKL) activates TAK1 mitogen-activated protein kinase kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6}, volume={22}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0036150184&partnerID=MN8TOARS}, DOI={10.1128/MCB.22.4.992-1000.2002}, abstractNote={AbstractThe receptor activator of NF-κB (RANK) and its ligand RANKL are key molecules for differentiation and activation of osteoclasts. RANKL stimulates transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation, and NF-κB through IκB kinase (IKK) activation. Tumor necrosis factor receptor-associated factor 6 (TRAF6) is essential for activation of these kinases. In the interleukin-1 signaling pathway, TAK1 MAPK kinase kinase (MAPKKK) mediates MAPK and IKK activation via interaction with TRAF6, and TAB2 acts as an adapter linking TAK1 and TRAF6. Here, we demonstrate that TAK1 and TAB2 participate in the RANK signaling pathway. Dominant negative forms of TAK1 and TAB2 inhibit NF-κB activation induced by overexpression of RANK. In 293 cells stably transfected with full-length RANK, RANKL stimulation facilitates the formation of a complex containing RANK, TRAF6, TAB2, and TAK1, leading to the activation of TAK1. Furthermore, in murine monocyte RAW 264.7 cells, dominant negative forms of TAK1 and TAB2 inhibit NF-κB activation induced by RANKL and endogenous TAK1 is activated in response to RANKL stimulation. These results suggest that the formation of the TRAF6-TAB2-TAK1 complex is involved in the RANK signaling pathway and may regulate the development and function of osteoclasts. We thank H. Nakano and M. Tsuda for materials, M. Tsuda, N. Sato, and N. Yanaka for helpful discussions, and M. Lamphier for critical reading of the manuscript.This work was supported by special grants to Advanced Research on Cancer from the Ministry of Education, Culture and Science of Japan (K.M.).}, number={4}, journal={Molecular and Cellular Biology}, author={Mizukami, J. and Takaesu, G. and Akatsuka, H. and Sakurai, H. and Ninomiya-Tsuji, J. and Matsumoto, K. and Sakurai, N.}, year={2002}, pages={992–1000} }
 @article{tanaka-hino_sagasti_hisamoto_kawasaki_nakano_ninomiya-tsuji_bargmann_matsumoto_2002, title={SEK-1 MAPKK mediates Ca2+ signaling to determine neuronal asymmetric development in Caenorhabditis elegans}, volume={3}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0036171872&partnerID=MN8TOARS}, DOI={10.1093/embo-reports/kvf001}, number={1}, journal={EMBO Reports}, author={Tanaka-Hino, M. and Sagasti, A. and Hisamoto, N. and Kawasaki, M. and Nakano, S. and Ninomiya-Tsuji, J. and Bargmann, C.I. and Matsumoto, K.}, year={2002}, pages={56–62} }
 @article{komatsu_shibuya_takeda_ninomiya-tsuji_yasui_miyado_sekimoto_ueno_matsumoto_yamada_2002, title={Targeted disruption of the Tab1 gene causes embryonic lethality and defects in cardiovascular and lung morphogenesis}, volume={119}, ISSN={["0925-4773"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0036895537&partnerID=MN8TOARS}, DOI={10.1016/S0925-4773(02)00391-X}, abstractNote={The transforming growth factor-beta (TGF-beta) superfamily consists of a group of secreted signaling molecules that perform important roles in the regulation of cell growth and differentiation. TGF-beta activated kinase-1 binding protein-1 (TAB1) was identified as a molecule that activates TGF-beta activated kinase-1 (TAK1). Recent studies have revealed that the TAB1-TAK1 interaction plays an important role in signal transduction in vitro, but little is known about the role of these molecules in vivo. To investigate the role of TAB1 during development, we cloned the murine Tab1 gene and disrupted it by homologous recombination. Homozygous Tab1 mutant mice died, exhibiting a bloated appearance with extensive edema and hemorrhage at the late stages of gestation. By histological examinations, it was revealed that mutant embryos exhibited cardiovascular and lung dysmorphogenesis. Tab1 mutant embryonic fibroblast cells displayed drastically reduced TAK1 kinase activities and decreased sensitivity to TGF-beta stimulation. These results indicate a possibility that TAB1 plays an important role in mammalian embryogenesis and is required for TAK1 activation in TGF-beta signaling.}, number={2}, journal={MECHANISMS OF DEVELOPMENT}, author={Komatsu, Y and Shibuya, H and Takeda, N and Ninomiya-Tsuji, J and Yasui, T and Miyado, K and Sekimoto, T and Ueno, N and Matsumoto, K and Yamada, G}, year={2002}, month={Dec}, pages={239–249} }
 @article{ishitani_kishida_hyodo-miura_ueno_yasuda_waterman_shibuya_moon_ninomiya-tsuji_matsumoto_2003, title={The TAK1-NLK mitogen-activated protein kinase cascade functions in the Wnt-5a/Ca<sup>2+</sup> pathway to antagonize Wnt/β-catenin signaling}, volume={23}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0037214344&partnerID=MN8TOARS}, DOI={10.1128/MCB.23.1.131-139.2003}, abstractNote={Wnt signaling controls a variety of developmental processes. The canonical Wnt/beta-catenin pathway functions to stabilize beta-catenin, and the noncanonical Wnt/Ca(2+) pathway activates Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). In addition, the Wnt/Ca(2+) pathway activated by Wnt-5a antagonizes the Wnt/beta-catenin pathway via an unknown mechanism. The mitogen-activated protein kinase (MAPK) pathway composed of TAK1 MAPK kinase kinase and NLK MAPK also negatively regulates the canonical Wnt/beta-catenin signaling pathway. Here we show that activation of CaMKII induces stimulation of the TAK1-NLK pathway. Overexpression of Wnt-5a in HEK293 cells activates NLK through TAK1. Furthermore, by using a chimeric receptor (beta(2)AR-Rfz-2) containing the ligand-binding and transmembrane segments from the beta(2)-adrenergic receptor (beta(2)AR) and the cytoplasmic domains from rat Frizzled-2 (Rfz-2), stimulation with the beta-adrenergic agonist isoproterenol activates activities of endogenous CaMKII, TAK1, and NLK and inhibits beta-catenin-induced transcriptional activation. These results suggest that the TAK1-NLK MAPK cascade is activated by the noncanonical Wnt-5a/Ca(2+) pathway and antagonizes canonical Wnt/beta-catenin signaling.}, number={1}, journal={Molecular and Cellular Biology}, author={Ishitani, T. and Kishida, S. and Hyodo-Miura, J. and Ueno, N. and Yasuda, J. and Waterman, M. and Shibuya, H. and Moon, R.T. and Ninomiya-Tsuji, J. and Matsumoto, K.}, year={2003}, pages={131–139} }
 @article{inoue_tateno_fujimura-kamada_takaesu_adachi-yamada_ninomiya-tsuji_irie_nishida_matsumoto_2001, title={A Drosophila MAPKKK, D-MEKK1, mediates stress responses through activation of p38 MAPK}, volume={20}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035477853&partnerID=MN8TOARS}, DOI={10.1093/emboj/20.19.5421}, number={19}, journal={EMBO Journal}, author={Inoue, H. and Tateno, M. and Fujimura-Kamada, K. and Takaesu, G. and Adachi-Yamada, T. and Ninomiya-Tsuji, J. and Irie, K. and Nishida, Y. and Matsumoto, K.}, year={2001}, pages={5421–5430} }
 @article{ono_ohtomo_sato_sugamata_suzuki_hisamoto_ninomiya-tsuji_tsuchiya_matsumoto_2001, title={An Evolutionarily Conserved Motif in the TAB1 C-terminal Region is Necessary for Interaction with and Activation of TAK1 MAPKKK}, volume={276}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035968214&partnerID=MN8TOARS}, DOI={10.1074/jbc.M102631200}, abstractNote={TAK1, a member of the MAPKKK family, is involved in the intracellular signaling pathways mediated by transforming growth factor β, interleukin 1, and Wnt. TAK1 kinase activity is specifically activated by the TAK1-binding protein TAB1. The C-terminal 68-amino acid sequence of TAB1 (TAB1-C68) is sufficient for TAK1 interaction and activation. Analysis of various truncated versions of TAB1-C68 defined a C-terminal 30-amino acid sequence (TAB1-C30) necessary for TAK1 binding and activation. NMR studies revealed that the TAB1-C30 region has a unique α-helical structure. We identified a conserved sequence motif, PYVDXA/TXF, in the C-terminal domain of mammalian TAB1, Xenopus TAB1, and itsCaenorhabditis elegans homolog TAP-1, suggesting that this motif constitutes a specific TAK1 docking site. Alanine substitution mutagenesis showed that TAB1 Phe-484, located in the conserved motif, is crucial for TAK1 binding and activation. The C. eleganshomolog of TAB1, TAP-1, was able to interact with and activate theC. elegans homolog of TAK1, MOM-4. However, the site in TAP-1 corresponding to Phe-484 of TAB1 is an alanine residue (Ala-364), and changing this residue to Phe abrogates the ability of TAP-1 to interact with and activate MOM-4. These results suggest that the Phe or Ala residue within the conserved motif of the TAB1-related proteins is important for interaction with and activation of specific TAK1 MAPKKK family members in vivo. TAK1, a member of the MAPKKK family, is involved in the intracellular signaling pathways mediated by transforming growth factor β, interleukin 1, and Wnt. TAK1 kinase activity is specifically activated by the TAK1-binding protein TAB1. The C-terminal 68-amino acid sequence of TAB1 (TAB1-C68) is sufficient for TAK1 interaction and activation. Analysis of various truncated versions of TAB1-C68 defined a C-terminal 30-amino acid sequence (TAB1-C30) necessary for TAK1 binding and activation. NMR studies revealed that the TAB1-C30 region has a unique α-helical structure. We identified a conserved sequence motif, PYVDXA/TXF, in the C-terminal domain of mammalian TAB1, Xenopus TAB1, and itsCaenorhabditis elegans homolog TAP-1, suggesting that this motif constitutes a specific TAK1 docking site. Alanine substitution mutagenesis showed that TAB1 Phe-484, located in the conserved motif, is crucial for TAK1 binding and activation. The C. eleganshomolog of TAB1, TAP-1, was able to interact with and activate theC. elegans homolog of TAK1, MOM-4. However, the site in TAP-1 corresponding to Phe-484 of TAB1 is an alanine residue (Ala-364), and changing this residue to Phe abrogates the ability of TAP-1 to interact with and activate MOM-4. These results suggest that the Phe or Ala residue within the conserved motif of the TAB1-related proteins is important for interaction with and activation of specific TAK1 MAPKKK family members in vivo. mitogen-activated protein kinase MAPK kinase kinase apoptosis signal-regulating kinase 1 C-terminal sequence of TAB1 hemagglutinin cytomegalovirus Gal4 transcription activation domain The mitogen-activated protein kinases (MAPKs)1 are a family of serine-threonine kinases that function in a wide variety of biological processes (1Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1407) Google Scholar). MAPKs are activated by phosphorylation on specific tyrosine and threonine residues by a family of dual-specificity protein kinase MAPK kinases. MAPK kinases are in turn activated by phosphorylation on serine and serine-threonine residues by the MAPK kinase kinases (MAPKKKs; Refs. 1Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1407) Google Scholar, 2Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). There are numerous MAPKKK-like proteins, including Raf, TAK1, Tpl2, apoptosis signal-regulating kinase 1 (ASK1), and the MEKK and mixed lineage kinase group of proteins (1Schaeffer H.J. Weber M.J. Mol. Cell. Biol. 1999; 19: 2435-2444Crossref PubMed Scopus (1407) Google Scholar, 2Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3666) Google Scholar). Several components that function upstream of the MAPKKK family have been identified. Ras GTPase functions upstream of the Raf MAPKKK and is itself activated by growth factors that signal through receptor protein-tyrosine kinases (3Kolch W. Biochem. J. 2000; 351: 289-305Crossref PubMed Scopus (1224) Google Scholar). Rac and Cdc42 GTPases can interact with the MEKK and mixed lineage kinase group of proteins (4Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (255) Google Scholar, 5Gerwins P. Blank J.L. Johnson G.L. J. Biol. Chem. 1997; 272: 8288-8295Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 6Tibbles L.A. Ing Y.L. Kiefer F. Chan J. Iscove N. Woodgett J.R. Lassam N.J. EMBO J. 1996; 15: 7026-7035Crossref PubMed Scopus (280) Google Scholar, 7Teramoto H. Coso O.A. Miyata H. Igishi T. Miki T. Gutkind J.S. J. Biol. Chem. 1996; 271: 27225-27228Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). TRAF2, an adaptor protein that functions in the tumor necrosis factor α signaling pathway, has been reported to bind MEKK1 and ASK1 (8Yuasa T. Ohno S. Kehrl J.H. Kyriakis J.M. J. Biol. Chem. 1998; 273: 22681-22692Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar, 9Nishitoh H. Saitoh M. Mochida Y. Takeda K. Nakano H. Rothe M. Miyazono K. Ichijo H. Mol. Cell. 1998; 2: 389-395Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). It has been demonstrated that TRAF2 acts on ASK1 after dissociation of ASK1 from its inhibitor thioredoxin (10Liu H. Nishitoh H. Ichijo H. Kyriakis J.M. Mol. Cell. Biol. 2000; 20: 2198-2208Crossref PubMed Scopus (450) Google Scholar). This interaction is likely to involve the regulation of ASK1 dimerization by reactive oxgen species (11Gotoh Y. Cooper J.A. J. Biol. Chem. 1998; 273: 17477-17482Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). In some signaling pathways, Ste20-like kinases have been implicated in the activation of MAPKKKs. For example, Raf-1 is phosphorylated and activated by p21 (Rac/Cdc42)-activated kinase (12King A.J. Sun H. Diaz B. Barnard D. Miao W. Bagrodia S. Marshall M.S. Nature. 1998; 396: 180-183Crossref PubMed Scopus (386) Google Scholar). Germinal center kinase functions upstream of MEKK1 in the tumor necrosis factor α signaling pathway, leading to Jun N-terminal kinase activation (8Yuasa T. Ohno S. Kehrl J.H. Kyriakis J.M. J. Biol. Chem. 1998; 273: 22681-22692Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar), and hematopoietic progenitor kinase and hematopoietic progenitor kinase /germinal center kinase-like kinase are involved in the activation of TAK1 (13Wang W. Zhou G. Hu M.C.-T. Yao Z. Tan T.H. J. Biol. Chem. 1997; 272: 22771-22775Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Thus, different mechanisms are involved in the activation of MAPKKKs in response to a variety of extracellular stimuli. To understand the mechanisms by which extracellular signals regulate the MAPK pathway, it is essential to characterize potential factors involved in MAPKKK activation. TAK1 is a member of the MAPKKK family and is activated by various cytokines, including transforming growth factor-β family ligands and interleukin 1 (14Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar, 15Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar). We have previously demonstrated that TAK1 functions in the transforming growth factor β signaling pathways in mammalian cells (15Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar). In early embryos of the amphibianXenopus, TAK1 also participates in mesoderm induction and patterning meditated by bone morphogenetic protein, another transforming growth factor β family ligand (16Shibuya H. Iwata H. Masuyama N. Gotoh Y. Yamaguchi K. Irie K. Matsumoto K. Nishida E. Ueno N. EMBO J. 1998; 17: 1019-1028Crossref PubMed Scopus (191) Google Scholar). Furthermore, we have recently demonstrated that TAK1 is involved in the interleukin 1 signaling pathway via its activation of two kinase cascades (14Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar), an MAP kinase cascade leading to Jun N-terminal kinase activation and an IκB kinase cascade that ultimately leads to nuclear factor κB activation. The TAB1 protein was isolated in a yeast two-hybrid screen as a specific partner of TAK1 (17Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (524) Google Scholar). When ectopically expressed together with TAB1, TAK1 phosphorylation and kinase activity are increased (18Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). A kinase-deficient mutant of TAK1 is not phosphorylated when co-expressed with TAB1, indicating that TAB1 promotes TAK1 autophosphorylation. Furthermore, mutation of a conserved serine residue (Ser-192) to alanine in the activation loop between kinase domains VII and VIII of TAK1 abolishes both phosphorylation and kinase activation by TAB1. These results suggest that ectopic expression of TAB1 activates TAK1 by promoting TAK1 autophosphorylation on Ser-192. However, the molecular mechanism for this activation remains to be elucidated. Homologs of TAK1 and TAB1, encoded by mom-4 andtap-1 genes, respectively, have been identified inCaenorhabditis elegans (19Meneghini M.D. Ishitani T. Carter J.C. Hisamoto N. Ninomiya-Tsuji J. Thorpe C.J. Hamill D.R. Matsumoto K. Bowerman B. Nature. 1999; 399: 793-797Crossref PubMed Scopus (235) Google Scholar). Biochemical analysis has revealed that MOM-4 can phosphorylate MKK6 MAPK kinase in vitro, and that MOM-4 kinase activity is promoted by binding of TAP-1 (19Meneghini M.D. Ishitani T. Carter J.C. Hisamoto N. Ninomiya-Tsuji J. Thorpe C.J. Hamill D.R. Matsumoto K. Bowerman B. Nature. 1999; 399: 793-797Crossref PubMed Scopus (235) Google Scholar). Thus, biochemical characterization of MOM-4 and TAP-1 suggests that these factors are similar to TAK1 and TAB1. Genetic analysis has shown that TAP-1 and MOM-4 regulate Wnt signaling inC. elegans via activation of the MAPK-like LIT-1. This is consistent with the observation that co-expression of TAK1 and TAB1 in mammalian cells can activate NEMO-like kinase, a mammalian homolog of LIT-1(20), and that the TAK1-NEMO-like kinase MAPK pathway negatively regulates Wnt signaling in mammalian cells. Thus, TAB1-TAK1 and TAP-1-MOM-4 appear to function analogously to regulate Wnt signaling pathways in mammalian cells and C. elegans, respectively. The C-terminal 68-amino acid portion of TAB1 was previously shown to be sufficient for TAK1 binding and activation (17Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (524) Google Scholar). To understand the mechanism for TAB1-induced activation of TAK1, we undertook a structural and functional analysis of this domain. We report here that the C-terminal region of TAB1 contains a unique α-helix structure. By analyzing a number of TAB1-related proteins, we identified a conserved motif, PYVDXA/TXF, that is present in the C-terminal domains. Our results suggest that this conserved region functions as a specific docking site for TAK1. Yeast two-hybrid analysis was performed as described previously (17Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (524) Google Scholar). For quantitative β-galactosidase assays, a liquid culture assay using theo-nitrophenyl-β-d-galactopyranoside substrate was carried out as described previously (21Bartel, P. L., Chien, C., Sternglanz, R., Fields, S., Cellular Interactions in Development: A Practical Approach, Hartley, D. A., 1993, 153, 179, Oxford University Press, Oxford, England.Google Scholar), and activity was expressed in Miller units (22Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar). For the ste11Δcomplementation assay, SY1984-P (his3Δ ste11Δ FUS1p::HIS3 STE7 P-368) was co-transformed with pNV11-TAK1 and the pGAD10 vector containing various TAB1 deletion mutants, and the transformants were streaked onto SC-His plates and incubated at 30 °C. TAB1 mutants were constructed by PCR-based mutagenesis using the appropriate synthetic oligonucleotides and the human TAB1 cDNA as a template. Resultant fragments were inserted into theXhoI-BamHI site of pGAD10 or into the mammalian expression vectors described below. All mutations were confirmed by sequencing. The samples for NMR analyses consisted of 6.8 mm C-terminal 30-amino acid sequence of TAB1 (TAB1-C30) in a solution of 40% trifluoroethanol, 55% H2O, and 5% D2O, pH 2.8. Under these conditions, the NMR spectra were well resolved. NMR measurements were performed using standard pulse sequences and phase cycling on a Bruker DMX-500 spectrometer. All two-dimensional NMR spectra were acquired in a phase-sensitive mode, using the time-proportional phase incrementation (23Marion D. Wuthrich K. Biochem. Biophys. Res. Commun. 1983; 113: 967-974Crossref PubMed Scopus (3524) Google Scholar) for quadrature detection in the t1dimension. NOESY spectra (24Jeener J. Meier B. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4845) Google Scholar, 25Macura S. Huang Y. Suter D. Ernst R.R. J. Magn. Reson. 1981; 43: 259-281Google Scholar) were recorded with mixing time of 200 ms at 300 K. TOCSY spectra were recorded using an MLEV-17 pulse scheme (26Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-359Google Scholar) with an isotropic mixing time of 50 ms. Spectra were referenced to H2O. Double-quantum filtered COSY experiments (27Rance M. Sorensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wuthrich K. Biochem. Biohys. Res. Commun. 1983; 117: 479-495Crossref PubMed Scopus (2597) Google Scholar) were recorded in an attempt to measure3JNH-H coupling constants (28Redfield A.G. Kunz S.D. J. Magn. Reson. 1975; 19: 250-254Google Scholar). The water proton signal was suppressed by low-power irradiation during the relaxation delay (2.0 s) of all experiments and during the mixing time of NOESY experiments. Data processing was performed on a Sun UltraSparc workstation using the program package NMRDraw and NMRPipe (29Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11638) Google Scholar). The strengths of the peaks were determined by peak height in the 200-ms NOESY spectrum. Peaks were classified as strong, medium, or weak. These classifications corresponded to the upper bounds of 2.8, 3.5, and 5.0 Å, respectively (30Clore G. Bruenger A.T. Karplus M. Gronenborn A.M. J. Mol. Biol. 1986; 205: 201-228Google Scholar). The upper limits of nonstereospecifically assigned methylene, methyl, and aromatic protons were adjusted using standard pseudoatom corrections (31Wuthrich K. Billeter M. Braun W. J. Mol. Biol. 1983; 169: 949-961Crossref PubMed Scopus (1007) Google Scholar). Three-dimensional structures were calculated on an SGI INDIGO2 workstation using simulated annealing and energy minimization protocols provided by X-PLOR 3.1 (32Bruenger A.T. Clore G.M. Gronenborn A.M. Karplus M. Proc. Natl. Acad. Sci. U. S. A. 1986; 74: 4130-4134Google Scholar, 33Bruenger A.T. X-POLOR Manual Version 3.1. Yale University, New Haven, CT1992Google Scholar). Anab initio-simulated annealing protocol was used to generate a set of 50 structures, on the basis of the experimentally derived distance and torsion angle constraints, starting from template structures with randomized angles and extended side chains (34Nilges M. Gronenborn A.M. Bruenger A.T. Clore G.M. Protein Eng. 1988; 2: 27-38Crossref PubMed Scopus (516) Google Scholar). 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with fetal calf serum (10%) at 37 °C and 5% CO2. pCMVHA and pCMVFlag vectors were constructed by inserting a hemagglutinin (HA) or Flag epitope sequence into pCMV, generating N-terminal HA or Flag epitope-tagged proteins, respectively. Mammalian expression vectors for N-terminal HA epitope-tagged TAK1 (pCMVHA-TAK1) and MOM-4 (pCMVHA-MOM-4) or N-terminal Flag epitope-tagged TAB1 (pCMVFlag-TAB1) and TAP-1 (pCMVFlag-TAP-1) were generated by inserting the cDNA fragments into the pCMVHA or pCMVFlag vectors, respectively. 293 cells (1 × 106) were plated in 10-cm dishes, transfected with various expression vectors, and incubated for 24–36 h. Cells were washed once with phosphate-buffered saline and lysed in 0.3 ml of 0.5% Triton X-100 lysis buffer containing 20 mm HEPES, pH 7.4, 150 mm NaCl, 12.5 mm β-glycerophosphate, 1.5 mm MgCl2, 2 mm EGTA, 10 mm NaF, 2 mm dithiothreitol, 1 mmsodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, and 20 μm aprotinin. Cellular debris was removed by centrifugation at 10,000 × g for 5 min. Proteins from cell lysates were incubated with the anti-HA rabbit polycolonal antibody Y-11 (Santa Cruz Biotechnologies) and 15 μl of protein G-Sepharose (Amersham Pharmacia Biotech). The beads were washed extensively with phosphate-buffered saline. For immunoblotting, the immunoprecipitates or whole cell lysates were resolved on SDS-polyacrylamide gel electrophoresis and transferred to Hybond-P membranes (Amersham Pharmacia Biotech). The membranes were immunoblotted with mouse monoclonal antibodies to HA, HA.11 (Babco), or Flag, M2 (Sigma). The bound antibody was visualized with horseradish peroxidase-conjugated antibodies to mouse IgG using the ECL Western blotting system (Amersham Pharmacia Biotech). Aliquots of immunoprecipitates were incubated in 10 μl of kinase buffer containing 10 mmHEPES, pH 7.4, 1 mm dithiothreitol, 5 mmMgCl2, and 5 μCi of [γ-32P]ATP at 25 °C for 2 min. Samples were resolved on SDS-polyacrylamide gel electrophoresis, and phosphorylated proteins were visualized by autoradiography. We have previously shown that the C-terminal 68-amino acid sequence of TAB1 (TAB1-C68) is sufficient for TAK1 interaction and activation (17Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (524) Google Scholar). To determine the region of TAB1-C68 responsible for its interaction with TAK1, we examined a series of N- and C-terminal TAB1-C68 truncation mutants in a yeast two-hybrid system. Various deletion mutants of TAB1-C68 were fused to the Gal4 transcription activation domain (GAD) and co-transformed with a plasmid encoding a LexA DNA binding domain-TAK1 chimeric protein. Interaction was detected by expression of a β-galactosidase reporter containing LexA binding sites in its promoter (Fig.1). Deletion of a serine-rich region within TAB1 (residues 437–460, TAB1-C45) had no effect on the binding of a TAB1 with TAK1. Thus, the serine-rich region of TAB1 is not required for interaction with TAK1, and the minimum TAB1 segment required for TAK1 binding includes residues 480–495. We have previously shown that a constitutively activated form of mammalian TAK1 can complement the yeast Ste11 MAPKKK in the pheromone-induced MAPK pathway (15Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar). However, expression of normal, full-length TAK1 fails to rescue the ste11Δ mutation. This suggests that yeast cells lack an endogenous activator of TAK1 (15Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar). Using this complementation system, various GAD-TAB1 constructs were tested for their ability to complement the ste11Δ mutation in the presence of TAK1. Co-expression of GAD-TAB1-C30 with TAK1 was found to rescue the Ste11 deficiency, whereas GAD-TAB1-C25 had no effect (Fig. 1). These results indicate that the serine-rich region of TAB1 is not required for either TAK1 binding or activation, whereas TAB1-C30 contains a minimal region sufficient for both TAK1 binding and activation. To determine the three-dimensional structure of the TAB1-C30 region, a peptide comprising residues 475–504 was synthesized and analyzed by NMR (Fig.2 A). For sequence-specific assignments and the identification of secondary structure elements in TAB1-C30, we used sequential nuclear Overhauser effect connectivities and the secondary Hα chemical shifts observed for TAB1-C30 in 40% trifluoroethanol, 55% H2O, and 5% D2O, pH 2.75. On the basis of the superposition of the 10 best-calculated structures, an α-helical region was identified between Tyr-481 and His-495. This region overlaps with the minimum region in TAB1 required for TAK1 binding (Fig. 1). However, the secondary structures of both the N-terminal (residues from Asp-475 to Phe-480) and C-terminal (residues from Asp-496 to Phe-504) regions were not well defined (Fig. 2 B). In a wheel projection of the α-helical region, we noticed the alignment of amino acids carrying large hydrophobic side chains with aromatic character on the same side of the wheel (Fig. 2 C). This structure may provide local apolar surfaces that facilitate the interaction of TAB1 with TAK1. To determine whether individual aromatic amino acids in the α-helix region between Tyr-481 and His-495 are essential for TAB1 interaction with TAK1, each aromatic amino acid in the TAB1-C68 region was replaced with alanine. TAK1 binding activity of wild-type and mutant TAB1-C68 was determined by a yeast two-hybrid protein assay (Fig. 3). Alanine substitution of Phe-484 markedly reduced TAK1 binding. In contrast, substitutions of Tyr-481, Phe-487, Trp-491, or His-495 with alanine had little effect on TAK1 binding. Alanine substitution of Tyr-488 partially reduced interaction with TAK1. Thus, this analysis implicates the Phe-484 residue within the α-helix as essential for TAK1 binding. We next examined whether the F484A mutation would affect the ability of full-length TAB1 to bind to TAK1. We constructed TAB1(F484A), replacing Phe-484 with Ala, and performed co-immunoprecipitation assays in mammalian cells. Expression vectors encoding Flag epitope-tagged TAB1 (Flag-TAB1) or TAB1(F484A) (Flag-TAB1(F484A)) were co-transfected into human embryonic kidney 293 cells along with an expression vector encoding HA epitope-tagged TAK1 (HA-TAK1). Cell lysates were immunoprecipitated with the anti-HA antibody, and co-precipitated TAB1 or TAB1(F484A) was detected by immunoblot analysis with the anti-Flag antibody. We found that the TAB1(F484A) mutation caused a marked reduction in TAK1 binding relative to wild-type TAB1 (Fig.4). Thus, the F484A mutation disrupts the association of full-length TAB1 with TAK1. We have previously shown that TAK1 has no kinase activity when ectopically expressed in mammalian cells alone but is activated when co-expressed with TAB1 (17Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (524) Google Scholar, 18Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). We therefore examined the effect of the TAB1(F484A) mutation on TAK1 activation. TAK1 immunoprecipitates were subjected to in vitro kinase assay using bacterially expressed MKK6 as an exogenous substrate (Fig. 4). As observed previously (17Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (524) Google Scholar, 18Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), TAK1 activity was not detected when TAK1 alone was ectopically expressed but was detected when TAK1 and TAB1 were expressed together. In contrast, TAK1 activity was significantly lower when the TAB1(F484A) mutant was co-expressed with TAK1. These results indicate that the Phe-484 residue in the TAB1 protein is important for both TAK1 binding and activation. TheC. elegans MOM-4 and TAP-1 proteins are structurally and functionally similar to the vertebrate TAK1 and TAB1 proteins, respectively (19Meneghini M.D. Ishitani T. Carter J.C. Hisamoto N. Ninomiya-Tsuji J. Thorpe C.J. Hamill D.R. Matsumoto K. Bowerman B. Nature. 1999; 399: 793-797Crossref PubMed Scopus (235) Google Scholar). For example, MOM-4 functions as an MAPKKK that can phosphorylate the MKK6 MAPK kinase in vitro, and TAP-1 can interact with and activate MOM-4. The present studies delineated an essential region required for TAK1 binding within the TAB1-C30 domain. This C-terminal region is homologous between the vertebrate TAB1 and the C. elegans TAP-1; in particular, a similar motif PYVDXA/TXF, is present in both (Fig.5). Therefore, it is likely that the TAK1/MOM-4 binding function of TAB1/TAP-1 is evolutionarily conserved. However, although mutation of Phe-484 to alanine abrogates the ability of mammalian TAB1 to associate with and activate TAK1, the corresponding site in the C. elegans TAP-1 protein is itself an alanine residue, Ala-364. To determine whether this alanine plays a role in MOM-4 binding and activation, we mutated Ala-364 to Phe. We transiently co-expressed HA epitope-tagged MOM-4 (HA-MOM-4) together with Flag epitope-tagged TAP-1 (Flag-TAP-1) or TAP-1(A364F) (Flag-TAP-1(A364F)) in 293 cells. Cell extracts were subjected to immunoprecipitation with anti-HA antibody, immunoblotting, and thenin vitro kinase assay (Fig. 4). Immunoblot analysis revealed that wild-type TAP-1 associated with MOM-4 but that the TAP-1(A364F) mutation abolished MOM-4 binding activity. When expressed alone, HA-MOM-4 had no kinase activity. However, co-expression with TAP-1 stimulated MOM-4 activity, whereas co-expression with TAP-1(A364F) did not. These results indicate that the Ala-364 residue in the C. elegans TAP-1 is important for both MOM-4 binding and activation. We next examined the species specificity between TAB1/TAP-1 and TAK1/MOM-4 by co-expression in 293 cells (Fig. 4). We found that neither mammalian Flag-TAB1 nor Flag-TAB1(F484A) associated with or activated C. elegans HA-MOM-4 kinase. In contrast, bothC. elegans TAP-1 and TAP-1(A364F) interacted with mammalian TAK1 kinase. Interestingly, although TAP-1 did not activate TAK1 kinase activity, TAP-1(A364F) did show weak stimulation of TAK1 autophosphorylation. Thus, activation of mammalian TAK1 requires a Phe residue in the C-terminal region of either TAB1 or TAP-1. The majority of protein kinases contain a number of conserved sequence motifs, in addition to the canonical ∼260-amino acid catalytic core, which have been suggested to be involved in substrate selection, regulation of catalytic activity, and cellular localization (35Johnson L.N. Noble M.E. Owen D.J. Cell. 1996; 85: 149-158Abstract Full Text Full Text PDF PubMed Scopus (1182) Google Scholar). In general, our understanding of the functions of these noncatalytic domains is poor, perhaps because there are few studies using purified, well-characterized proteins. In the present study, we examined TAK1, a member of the MAPKKK family. TAK1 is inactive until activated by TAB1. The activation of TAK1 is regulated by protein-protein interactions and protein modifications such as phosphorylation. Recently, we have shown that TAB1-induced activation of TAK1 requires autophosphorylation of TAK1 at Ser-192 in the kinase activation loop. Furthermore, it has been reported that the C-terminal region of TAB1, consisting of residues 480–504, is sufficient for association with and activation of TAK1 (36Sakurai H. Miyoshi H. Mizukami J. Sugita T. FEBS Lett. 2000; 474: 141-145Crossref PubMed Scopus (142) Google Scholar). In the present study, we characterized in detail the region in TAB1 responsible for binding to TAK1. First, a structural analysis of the TAB1-C30 peptide by NMR study reveals a unique α-helix structure, characterized by the alignment of aromatic residues on one side of the helix. Yeast two-hybrid analyses of wild-type, alanine-substituted, and deletion mutants of TAB1 show that the aromatic Phe-484 residue in the α-helical domain is critical for TAK1 binding. An evolutionarily conserved consensus core sequence required for TAK1 binding, PYVDXA/TXF, was identified within the helix domain of TAB1. High-resolution structural studies will be required to define the specific functional role of each amino acid in the core motif important for TAK1 binding. As shown here, TAK1 binding activity can be localized to a small region in the TAB1 C-terminal domain and can be ablated by a single point mutation. These features of the TAB1-TAK1 interaction suggest that small peptides or peptidomimetics that disrupt the interaction could be used to inhibit TAK1-dependent cellular functions.}, number={26}, journal={Journal of Biological Chemistry}, author={Ono, K. and Ohtomo, T. and Sato, S. and Sugamata, Y. and Suzuki, M. and Hisamoto, N. and Ninomiya-Tsuji, J. and Tsuchiya, M. and Matsumoto, K.}, year={2001}, pages={24396–24400} }
 @article{qian_commane_ninomiya-tsuji_matsumoto_li_2001, title={IRAK-mediated Translocation of TRAF6 and TAB2 in the Interleukin-1-induced Activation of NFκB}, volume={276}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035834672&partnerID=MN8TOARS}, DOI={10.1074/jbc.M102262200}, abstractNote={The interleukin-1 (IL-1) receptor-associated kinase (IRAK) is required for the IL-1-induced activation of nuclear factor κB and c-Jun N-terminal kinase. The goal of this study was to understand how IRAK activates the intermediate proteins TRAF6, TAK1, TAB1, and TAB2. When IRAK is phosphorylated in response to IL-1, it binds to the membrane where it forms a complex with TRAF6; TRAF6 then dissociates and translocates to the cytosol. The membrane-bound IRAK similarly mediates the IL-1-induced translocation of TAB2 from the membrane to the cytosol. Different regions of IRAK are required for the translocation of TAB2 and TRAF6, suggesting that IRAK mediates the translocation of each protein separately. The translocation of TAB2 and TRAF6 is needed to form a TRAF6-TAK1-TAB1-TAB2 complex in the cytosol and thus activate TAK1. Our results show that IRAK is required for the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2. The phosphorylation of these three proteins correlates strongly with the activation of nuclear factor κB but is not necessary to activate c-Jun N-terminal kinase. The interleukin-1 (IL-1) receptor-associated kinase (IRAK) is required for the IL-1-induced activation of nuclear factor κB and c-Jun N-terminal kinase. The goal of this study was to understand how IRAK activates the intermediate proteins TRAF6, TAK1, TAB1, and TAB2. When IRAK is phosphorylated in response to IL-1, it binds to the membrane where it forms a complex with TRAF6; TRAF6 then dissociates and translocates to the cytosol. The membrane-bound IRAK similarly mediates the IL-1-induced translocation of TAB2 from the membrane to the cytosol. Different regions of IRAK are required for the translocation of TAB2 and TRAF6, suggesting that IRAK mediates the translocation of each protein separately. The translocation of TAB2 and TRAF6 is needed to form a TRAF6-TAK1-TAB1-TAB2 complex in the cytosol and thus activate TAK1. Our results show that IRAK is required for the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2. The phosphorylation of these three proteins correlates strongly with the activation of nuclear factor κB but is not necessary to activate c-Jun N-terminal kinase. interleukin-1 N-acetyl-leucine-leucine-methionine death domain of interleukin-1 receptor-associated kinase IRAK, interleukin-1 receptor-associated kinase c-Jun N-terminal kinase kinase domain of interleukin-1 receptor-associated kinase deletion mutant of KD nuclear factor-κB nuclear factor-κB-inducing kinase polyacrylamide gel electrophoresis transforming growth factor-β-activated kinase 1-binding protein 1/2 transforming growth factor-β-activated kinase 1 tumor necrosis factor receptor-associated factor 6 domain of unknown function of interleukin-1 receptor-associated kinase deletion mutant of UD Interleukin-1 (IL-1)1plays a central role in mediating a variety of inflammatory responses, exerting its biological influences mainly by activating the transcription of effector genes in target cells (1Dinarello C.A. Blood. 1996; 87: 2095-2147Crossref PubMed Google Scholar). The transcription factors activated by IL-1 include nuclear factor-κB (NFκB), activating protein 1, and activating transcription factor (2Barnes P.J. Karin M. N. Engl. J. Med. 1997; 336: 1066-1071Crossref PubMed Scopus (4256) Google Scholar, 3O'Neill L.A. Biochim. Biophys. Acta. 1995; 1266: 31-44Crossref PubMed Scopus (119) Google Scholar, 4O'Neill L.A. Biochem. Soc. Trans. 1997; 25: 295-302Crossref PubMed Scopus (25) Google Scholar). IL-1-mediated signaling begins when IL-1 binds to the receptor complex, composed of the IL-1 receptor (IL-1R) and its accessory protein (5Greenfeder S.A. Nunes P. Kwee L. Labow M. Chizzonite R.A. Ju G. J. Biol. Chem. 1995; 270: 13757-13765Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 6Huang J. Gao X. Li S. Cao Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12829-12832Crossref PubMed Scopus (194) Google Scholar, 7Korherr C. Hofmeister R. Wesche H. Falk W. Eur. J. Immunol. 1997; 27: 262-267Crossref PubMed Scopus (153) Google Scholar). The cytosolic proteins MyD88 (8Lord K.A. Hoffman-Liebermann B. Liebermann D.A. Oncogene. 1990; 5: 1095-1097PubMed Google Scholar, 9Wesche H. Henzel W.J. Shillinglaw W. Li S. Cao Z. Immunity. 1997; 7: 837-847Abstract Full Text Full Text PDF PubMed Scopus (915) Google Scholar, 10Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Abstract Full Text Full Text PDF PubMed Scopus (1704) Google Scholar) and Tollip (11Burns K. Clatworthy J. Martin L. Martinon F. Plumpton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (445) Google Scholar) are then recruited to the receptor complex, where they function as adaptors, recruiting IL-1 receptor-associated kinase (IRAK) in turn. IRAK, a serine-threonine kinase, is phosphorylated at the receptor complex and then presumably dissociates from that complex to interact with tumor necrosis factor receptor-associated factor 6 (TRAF6) (12Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar, 13Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1117) Google Scholar, 14Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar, 15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar, 16Lomaga M.A. Yeh W.-C. Sarosi I. Duncan G.S. Furlonger C. Ho A. Morony S. Capparelli C. Van G. Kaufman S. van der Heiden A. Itie A. Wakeham A. Khoo W. Sasaki T. Cao Z. Penninger J.M. Paige C.J. Lacey D.L. Dunstan C.R. Boyle W.J. Goeddel D.V. Mak T.W. Genes Dev. 1999; 13: 1015-1024Crossref PubMed Scopus (1073) Google Scholar). Phosphorylated IRAK is eventually ubiquitinated and degraded (17Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). Transforming growth factor-β-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, and two proteins that bind to it, transforming growth factor-β-activated kinase 1 binding protein 1 and 2 (TAB1 and TAB2), have recently been implicated in IL-1 signaling (18Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1014) Google Scholar, 19Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). TAB2 is membrane-bound in untreated cells but translocates to the cytosol upon stimulation with IL-1, where it functions as an adaptor, linking TRAF6 to TAK1 and TAB1, thereby activating TAK1 (19Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). Some investigators (20Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1160) Google Scholar) have suggested that activated TAK1 triggers the NFκB-inducing kinase (NIK) and IκB kinase cascade, leading to NFκB activation. More recently, studies on NIK-deficient mice have revealed that NIK is required for lymphotoxin-β signaling but is dispensable for IL-1 and tumor necrosis factor-α signaling (21Yin L. Wu L. Wesche H. Arthur C.D. White J.M. Goeddel D.V. Schreiber R.D. Science. 2001; 291: 2162-2165Crossref PubMed Scopus (347) Google Scholar, 22Matsushima A. Kaisho T. Rennert P.D. Nakano H. Kurosawa K. Uchida D. Takeda K. Akira S. Matsumoto M.J. J. Exp. Med. 2001; 193: 631-636Crossref PubMed Scopus (177) Google Scholar). In light of these contrasting results, it remains an open question whether a NIK-like molecule (not NIK itself) is involved in the IL-1 signaling pathway. Activated IκB kinase phosphorylates the inhibitory IκB proteins, which are then degraded, releasing NFκB, which translocates to the nucleus, where it activates transcription (23DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1900) Google Scholar, 24Mercurio F. Zhu H. Murray B.W. Shevchenko A. Bennett B.L. Li J. Young D.B. Barbosa M. Mann M. Manning A. Rao A. Science. 1997; 278: 860-866Crossref PubMed Scopus (1841) Google Scholar, 25Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar, 26Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (845) Google Scholar, 27Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1065) Google Scholar, 28Yamaoka S. Courtois G. Bessia C. Whiteside S.T. Weil R. Agou F. Kirk H.E. Kay R.J. Israel A. Cell. 1998; 93: 1231-1240Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar, 29Zandi E. Rothwarf D.M. Delhase M. Hayakawa M. Karin M. Cell. 1997; 91: 243-252Abstract Full Text Full Text PDF PubMed Scopus (1575) Google Scholar, 30Li Q. Lu Q. Hwang J.Y. Buscher D. Lee K.-F. Izpisua-Belmonte J.C. Verma I.M. Genes Dev. 1999; 13: 1322-1328Crossref PubMed Scopus (416) Google Scholar, 31Li Q. Estepa G. Memet S. Israel A. Verma I.M. Genes Dev. 2000; 14: 1729-1733PubMed Google Scholar, 32Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Crossref PubMed Scopus (708) Google Scholar, 33Rudolph D. Yeh W.-C. Wakeham A. Rudolph B. Nallainathan D. Potter J. Elia A.J. Mak T.W. Genes Dev. 2000; 14: 854-862PubMed Google Scholar). Activated TAK1 has also been implicated in the IL-1-induced activation of c-Jun N-terminal kinase (JNK) and other mitogen-activated protein kinases (18Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1014) Google Scholar), leading to the phosphorylation and activation of activating transcription factor and activating protein 1, thereby also activating transcription. By taking a genetic approach to study IL-1 signaling pathways, we used random mutagenesis to generate IL-1-unresponsive cell lines lacking specific components of the pathways, and we have isolated the mutant cell line I1A, which lacks both IRAK protein and mRNA (14Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar, 15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar). IRAK is a multidomain protein (12Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar, 17Yamin T.T. Miller D.K. J. Biol. Chem. 1997; 272: 21540-21547Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar) containing an N-terminal death domain (DD, residues 1–103), followed by a domain of unknown function (UD, residues 104–198), a kinase domain (KD, residues 199–522), and a two-part C-terminal domain, also of unknown function (residues 523–618 for C1 and residues 619–712 for C2). IRAK-deficient I1A cells have been used effectively to study the function of IRAK in IL-1-dependent signaling (14Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar, 15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar). Neither NFκB nor JNK is activated in IL-1-treated I1A cells, but these responses are restored in I1A-IRAK cells, indicating that IRAK is required for both. To address the role of the kinase activity of IRAK, its ATP-binding site was mutated (K239A) or the entire kinase domain was deleted (dKD). IRAK-K239A and IRAK-dKD complemented all defects in IL-1-dependent signaling in mutant I1A cells, indicating that the kinase activity of IRAK is not required for these functions (14Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar, 15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar). K239A and dKD were still phosphorylated in I1A cells upon IL-1 stimulation, showing that IRAK must become phosphorylated by another kinase. Analysis of other IRAK deletion mutants showed that both the DD and C1C2 domains of IRAK are required for both NFκB and JNK activation but that the UD domain was necessary only for NFκB, not JNK, activation (15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar). Furthermore, IL-1-induced phosphorylation of IRAK is required to activate NFκB but not JNK (15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar). Taken together, these results strongly suggest that the IL-1-induced signaling pathways leading to NFκB and JNK activation diverge at or upstream of IRAK. It is clear that IRAK plays a critical role in IL-1 signaling. We now address the mechanisms of the IRAK-mediated activation of the downstream signaling proteins TRAF6, TAK1, TAB1, and TAB2. Previous studies with IRAK-deficient I1A cells have revealed that IRAK is required for the activation of TAK1, the translocation of TAB2 from the membrane to the cytosol upon IL-1 stimulation, and the formation of a TRAF6-TAK1-TAB2 complex (34Takaesu G. Ninomiya-Tsuji J. Kishida S. Li X. Stark G.R. Matsumoto K. Mol. Cell. Biol. 2001; 21: 2475-2484Crossref PubMed Scopus (159) Google Scholar). Here, we report that stimulation with IL-1 also leads to the translocation of TRAF6 from the membrane to the cytosol and that this process requires IRAK. We have identified the domains of IRAK required for the translocation of TAB2 and TRAF6, and we propose that IRAK mediates those events separately through protein-protein interactions that are modulated by phosphorylation. Recombinant human IL-1β was provided by the NCI, National Institutes of Health. Antibodies against IRAK and TRAF6 were a kind gift from Dr. Zhaodan Cao (Tularik, South San Francisco, CA). Anti-ubiquitin antibody was purchased from StressGen (Victoria, British Columbia, Canada). 293-TK/Zeo and I1A cells (14Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar) were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, penicillin G (100 μg/ml), and streptomycin (100 μg/ml).N-Acetyl-leucine-leucine-methionine (ALLM) was obtained from Calbiochem. IRAK deletion fragments, generated by polymerase chain reaction using IRAK-K239A as a template, were cloned into an expression vector (15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar) driven by the thymidine kinase gene promoter. For stable transfections, 2 × 105 cells seeded onto a 10-cm plate were co-transfected the following day by the calcium phosphate method with 10 μg of each expression vector and 1 μg of pBabePuro. After 48 h, the cells were selected with 1 μg/ml puromycin until clones appeared. Cells at 70% confluence, untreated or treated with IL-1 (100 IU/ml) for various times, were resuspended in 10 packed cell volumes of ice-cold hypotonic buffer (10 mm HEPES, pH 7.4, 1.5 mm MgCl2, 10 mm KCl, 0.2 mm phenylmethylsulfonyl fluoride, and 0.5 mm dithiothreitol) and homogenized on ice with 30 strokes of a Dounce homogenizer. Unlysed cells, nuclei, and cell debris were pelleted by centrifugation at 1000 ×g for 5 min. Soluble (supernatant, S100) and particulate (pellet, P100) fractions were generated by centrifugation at 100,000 × g for 1 h. Samples were separated by SDS-PAGE, and Western blot transfers were analyzed with different antibodies. Membrane fractions used for immunoprecipitation experiments were lysed in lysis buffer containing 0.5% Triton X-100, 20 mm HEPES, pH 7.4, 150 mm NaCl, 12.5 mm β-glycerophosphate, 1.5 mmMgCl2, 2 mm EGTA, 10 mm NaF, 2 mm dithiothreitol, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, and 20 μmaprotinin. Cell extracts were incubated with 1 μl of anti-IRAK or anti-TRAF6 polyclonal antibody (from Zhaodan Cao, Tularik, South San Francisco, CA) or preimmune serum for 4 h, followed by a 1-h incubation with 50 μl of packed protein A-Sepharose beads (pre-washed and resuspended in phosphate-buffered saline at a 1:1 ratio). After incubation, the beads were washed five times with lysis buffer, followed by Western analysis with anti-IRAK or anti-TRAF6 antibodies. It has been reported that IL-1 stimulates translocation of TAB2 from the membrane to the cytosol, where it mediates the IL-1-dependent association of TAK1 with TRAF6 (19Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). We found that TRAF6 also translocates from the membrane to the cytosol upon IL-1 stimulation. To examine the subcellular distribution of the IL-1 signaling proteins, wild-type 293 cells, untreated or treated with IL-1, were fractionated into membranes and cytosol, which were then analyzed with antibodies against IRAK, TRAF6, TAK1, TAB1, and TAB2. The IL-1 receptor and α-tubulin were used as markers for the membrane and cytosol fractions, respectively. Whereas unmodified IRAK was found in both membrane and cytosol fractions before IL-1 treatment, the phosphorylated and ubiquitinated IRAK induced by stimulation with IL-1 was found only in the membrane fraction (Fig.1 A). The IL-1-induced phosphorylation of IRAK was confirmed by [32P]orthophosphate labeling of the wild-type 293 cells untreated or treated with IL-1, followed by immunoprecipitation with anti-IRAK antibody and analysis by SDS-PAGE (Fig. 1 B). Since the IRAK deletion mutant dUD, lacking the N-proximal undetermined region, is not phosphorylated upon IL-1 stimulation, IRAK-deficient I1A cells transfected with dUD were used as a negative control (Fig.1 B). To confirm that IRAK is ubiquitinated upon IL-1 stimulation, cell extracts from 293 cells untreated or treated with IL-1 were immunoprecipitated with anti-IRAK antibody followed by Western analysis with anti-ubiquitin antibody (Fig. 1 C). As shown in Fig. 1 A, IRAK gradually disappeared from the cytosol fraction after stimulation with IL-1; this change probably resulted from the signal-induced modification of IRAK and its membrane localization after being modified. The modified IRAK in the membrane fraction was eventually degraded 6 h after stimulation. 2Z. Jiang, Y. Qian, and X. Li, unpublished data. TAB2 and TRAF6, mostly in the membrane fraction in untreated cells, translocated to the cytosol upon stimulation with IL-1 (Fig. 1 A). The Scion Image 1.62C program was used to quantitate the amount of TRAF6 and TAB2 in the cytosol before and after IL-1 stimulation. From the averages of five experiments, we observed a 5.6-fold increase in TRAF6 and an 8.5-fold increase in TAB2 in the cytosol 30 min after treatment with IL-1. TAK1 and TAB1, on the other hand, were found mostly in the cytosol before and after stimulation (Fig. 1 A). By using I1A cells, IRAK was shown to be required for the translocation of TAB2 from the membrane to the cytosol (34Takaesu G. Ninomiya-Tsuji J. Kishida S. Li X. Stark G.R. Matsumoto K. Mol. Cell. Biol. 2001; 21: 2475-2484Crossref PubMed Scopus (159) Google Scholar). As shown above, IL-1 also induced the translocation of TRAF6, and similar to findings with TAB2, the TRAF6 translocation was abolished in I1A cells (Fig. 2). Overexpression of wild-type IRAK or IRAK-K239A in I1A cells resulted in some constitutive TRAF6 translocation. Stimulation with IL-1 induced this translocation further (Figs. 2 and3), indicating that the kinase activity of IRAK is not important for the translocation of TRAF6.Figure 3Domains of IRAK required for the IL-1-induced translocation of TRAF6 and TAB2. Membrane and cytosolic fractions were prepared from untreated or IL-1-treated (100 IU/ml) I1A cells transfected with IRAK-K239A or IRAK deletion mutants. These fractions were probed with antibodies against TRAF6 and TAB2 after Western blot transfer. This experiment was repeated three times with consistent results. P100, particulate fraction; S100, soluble fraction.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To identify which domains of IRAK are required for the translocation of TRAF6 and TAB2, we studied the subcellular distribution of these proteins in I1A cells transfected with IRAK deletion constructs. Previous studies (14Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar) have shown that overexpression of wild-type IRAK in transfected I1A cells leads to its autophosphorylation, whereas IRAK-K239A is only phosphorylated upon stimulation. The deletion mutant lacking the kinase domain behaved like IRAK-K239A in mediating the translocation of TRAF6 and TAB2 (data not shown). To avoid possible complications from autophosphorylation, the other IRAK deletion constructs were generated from IRAK-K239A. Deletion of DD or C1C2 abolished the ability of IRAK to mediate both the IL-1-induced translocation of TRAF6 and TAB2; deletion of UD was only that of TRAF6 (Fig. 3). The IL-1-induced translocation of TAB2 was not affected in I1A-dUD cells (Fig. 3). These results show that different regions of IRAK are required for the translocation of TRAF6 and TAB2, suggesting that IRAK mediates their translocation by separate paths. How does IRAK mediate the translocation of TRAF6? Upon stimulation with IL-1, IRAK was phosphorylated and the modified IRAK was found only in the membrane fraction (Fig. 1). TRAF6 was found mostly in the membrane fraction before stimulation and was translocated to the cytosol after IL-1 treatment. Furthermore, previous studies (13Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1117) Google Scholar) have shown that IRAK interacts with TRAF6 upon IL-1 stimulation. Therefore, it is possible that phosphorylated IRAK, induced by IL-1-stimulation, interacts with TRAF6 at the membrane to facilitate the translocation of TRAF6 to the cytosol, where TRAF6 interacts with downstream proteins. To test this model, membrane fractions prepared from wild-type cells untreated or treated with IL-1 were resuspended in lysis buffer, immunoprecipitated with anti-IRAK, and probed with anti-TRAF6 or vice versa. An IRAK-TRAF6 complex was indeed formed in these membrane fractions after IL-1 stimulation (Fig.4 A). Cytosolic fractions were also obtained from wild-type cells untreated or treated with IL-1. Anti-TRAF6 was used to immunoprecipitate these cytosolic fractions, which were analyzed with antibodies against TAB2, TAK1, TAB1, and IRAK after Western blot transfer. TRAF6 formed a complex with TAB2, TAK1, and TAB1 in the IL-1-treated cytosolic fractions (Fig. 4 B). IRAK was not detected in the cytosol, indicating that the IRAK-TRAF6 complex dissociates before TRAF6 is translocated. The majority of the IRAK-TRAF6 complex was dissociated at 10 min after IL-1 stimulation (Fig. 4 A), which correlates with the time of translocation of TRAF6 to the cytosol (Fig. 1 A) and formation of the TRAF6-TAK1-TAB1-TAB2 complex in the cytosol (Fig. 4 B). We then examined whether the IL-1-induced degradation of IRAK plays a role in the dissociation of the IRAK-TRAF6 complex and the subsequent translocation of TRAF6 to the cytosol. Wild-type 293 cells were treated with IL-1 for various times (10 min to 4 h) with or without the presence of protease inhibitor ALLM, followed by Western blot analysis with an anti-IRAK antibody. As shown in Fig.5 A, ALLM inhibited the IL-1-induced degradation of IRAK. However, the inhibition of IRAK degradation had no effect on the IL-1-induced formation or dissociation of the IRAK-TRAF6 complex (data not shown). Likewise, treatment with ALLM did not affect the IL-1-induced IRAK-mediated translocation of TAB2 and TRAF6 (Fig. 5 B). These results suggest that the release of TRAF6 and TAB2 from IRAK is probably not through the degradation of IRAK. Stimulation with IL-1 leads to the activation of TAK1 (18Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1014) Google Scholar) and to the phosphorylation of TAK1, TAB1, and TAB2 (19Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 35Kishimito K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). The activation of TAK1 leads to the autophosphorylation of TAK1 and TAB1, but a protein kinase that functions upstream of TAK1 is required for the phosphorylation of TAB2 (19Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). We have recently shown that IRAK is required for the IL-1-induced activation of TAK1 (34Takaesu G. Ninomiya-Tsuji J. Kishida S. Li X. Stark G.R. Matsumoto K. Mol. Cell. Biol. 2001; 21: 2475-2484Crossref PubMed Scopus (159) Google Scholar). In the current study, we examined the additional role of IRAK in the IL-1-induced activation of TAK1 and phosphorylation of TAK1, TAB1, and TAB2. The IL-1-induced TAK1 kinase activity was abolished in IRAK-null I1A cells (Fig. 6 A). Although TAK1 was constitutively activated in I1A cells transfected with wild-type IRAK, it was only activated upon stimulation in I1A cells transfected with the IRAK kinase-dead mutant (K293A). Western blot analysis revealed that the TAK1, TAB1, and TAB2 proteins from wild-type cells migrated more slowly in SDS-PAGE when cells were treated with IL-1, indicating that they are phosphorylated (Fig. 6 B) (19Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, 35Kishimito K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). The IL-1-induced phosphorylation of these proteins was abolished in IRAK-null I1A cells (Fig. 6 B). Interestingly, whereas TAK1, TAB1, and TAB2 were phosphorylated constitutively in I1A cells transfected with wild-type IRAK, they were also phosphorylated in I1A cells transfected with the kinase-dead K239A upon IL-1 stimulation (Fig. 6 B). These results show that, although IRAK is required for the IL-1-induced activation of TAK1 and phosphorylation of TAK1, TAB1, and TAB2, the kinase activity of IRAK is not required for these events. Therefore, IRAK is not the kinase that phosphorylates TAB2. We then investigated the cause for the constitutive activation of TAK1 and phosphorylation of TAK1, TAB1, and TAB2 in I1A-IRAK cells. IRAK was constitutively autophosphorylated in I1A-IRAK cells, whereas IRAK-K239A was only phosphorylated upon IL-1 stimulation (Fig. 6 B). The phosphorylated forms of IRAK were confirmed by both [32P]orthophosphate labeling (Fig. 1 B) and treatment with calf intestinal phosphatase (14Li X. Commane M. Burns C. Vithalani K. Cao Z. Stark G.R. Mol. Cell. Biol. 1999; 19: 4643-4652Crossref PubMed Scopus (187) Google Scholar). The phosphorylated IRAK in I1A-IRAK cells was capable of forming a constitutive complex with TRAF6, whereas IRAK-K239A only formed a complex with TRAF6 upon IL-1 stimulation (Fig. 7). Therefore, it is possible that the phosphorylated IRAK is responsible for the constitutive phosphorylation of TAK1, TAB1, and TAB2 in I1A-IRAK cells through its interaction with TRAF6, thereby activating downstream signaling events. We examined the phosphorylation of TAK1, TAB1, and TAB2 in I1A cells transfected with different IRAK deletion constructs. Since the expression of wild-type IRAK in I1A cells leads to the constitutive phosphorylation of TAK1, TAB1, and TAB2, deletion constructs derived from the IRAK-K239A mutant were used. When we tested for IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 in the transfected I1A cells, we found that deletion of the DD, UD, or C1C2 regions completely abolished it, whereas deletion of the kinase domain of IRAK had no effect (Fig. 8). Therefore, although the kinase domain of IRAK is dispensable, both the N-terminal and C-terminal regions are required for the activation of TAK1, TAB1, and TAB2. This conclusion was supported further by results obtained with the truncated protein DD + UD + C1, in which the N-terminal domains (DD and UD) were fused with a part of the C-terminal region (C1). DD + UD + C1 behaved like full-length IRAK-K239A in conferring IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 in transfected I1A cells (Fig. 8). As summarized in Fig. 9, the IRAK deletion mutants that confer IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 in transfected I1A cells are themselves phosphorylated upon IL-1 stimulation (Fig. 8) and form a signal-dependent complex with TRAF6 (15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar), confirming the correlation between IRAK phosphorylation and phosphorylation of TAK1, TAB1, and TAB2. Fig. 9also shows that the domains of IRAK (DD, UD, and C1C2) that are required for the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 are also necessary for the IL-1-induced activation of NFκB (15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar), indicating that the phosphorylation of TAK1, TAB1, and TAB2 correlates with NFκB activation. However, the deletion mutant dUD, which fails to activate NFκB, TAK1, TAB1, and TAB2, still activates JNK (15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar) (Fig. 9), implying that the activation of TAK1, TAB1, and TAB2 is not necessary for the IL-1-induced activation of JNK. According to current models (10Adachi O. Kawai T. Takeda K. Matsumoto M. Tsutsui H. Sakagami M. Nakanishi K. Akira S. Immunity. 1998; 9: 143-150Abstract Full Text Full Text PDF PubMed Scopus (1704) Google Scholar, 11Burns K. Clatworthy J. Martin L. Martinon F. Plumpton C. Maschera B. Lewis A. Ray K. Tschopp J. Volpe F. Nat. Cell Biol. 2000; 2: 346-351Crossref PubMed Scopus (445) Google Scholar, 12Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar, 13Cao Z. Xiong J. Takeuchi M. Kurama T. Goeddel D.V. Nature. 1996; 383: 443-446Crossref PubMed Scopus (1117) Google Scholar, 19Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar), upon stimulation with IL-1, IRAK is recruited to the receptor complex, where it is phosphorylated. Phosphorylated IRAK then dissociates from the receptor, followed by its interaction with TRAF6 to activate the downstream signaling components. As summarized in Fig.10, we report here that phosphorylated IRAK, induced by IL-1 treatment, stays at the membrane, where it forms a complex with TRAF6; TRAF6 then dissociates and translocates to the cytosol to form a complex with TAK1-TAB1-TAB2. Previous studies (34Takaesu G. Ninomiya-Tsuji J. Kishida S. Li X. Stark G.R. Matsumoto K. Mol. Cell. Biol. 2001; 21: 2475-2484Crossref PubMed Scopus (159) Google Scholar) have indicated that IRAK also mediates the IL-1-induced translocation of TAB2 from the membrane to the cytosol. We found that different regions of IRAK are required for the translocation of TAB2 and TRAF6, suggesting that IRAK mediates these two events separately. The domains of IRAK required for the IL-1-induced translocation of TRAF6 are the same as those necessary for the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 and activation of NFκB (15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar) (Fig. 9), revealing a tight correlation between the translocation of TRAF6 and the activation of downstream signaling. It is likely that both TAB2 and TRAF6 need to be translocated to the cytosol for the TRAF6-TAK1-TAB1-TAB2 complex to form and for these proteins to be activated. Furthermore, our studies show that the phosphorylation of TAK1, TAB1, and TAB2 correlates tightly with IL-1-induced NFκB activation but is not necessary for JNK activation. We propose that IRAK mediates the IL-1-induced translocation of TRAF6 and TAB2, which leads to the formation of the TRAF6-TAK1-TAB1-TAB2 complex in the cytosol, the activation of TAK1, and the phosphorylation of TAK1, TAB1, and TAB2, resulting eventually in IL-1-induced NFκB activation (Fig. 10). At present, it is still not clear exactly how IRAK mediates the translocation of TRAF6 and TAB2. The translocation is likely to occur through the interaction of IRAK with and dissociation from these two proteins. The phosphorylation of IRAK seems to play an important role in its interaction with them. In wild-type cells, both TAB2 and TRAF6 interact with phosphorylated IRAK upon IL-1 stimulation (12Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (768) Google Scholar, 32Hu Y. Baud V. Delhase M. Zhang P. Deerinck T. Ellisman M. Johnson R. Karin M. Science. 1999; 284: 316-320Crossref PubMed Scopus (708) Google Scholar). Furthermore, the autophosphorylation of IRAK in untreated I1A-IRAK cells leads to its constitutive interaction with TRAF6 (Fig. 7). Therefore, it is possible that TRAF6 forms a transient complex with phosphorylated IRAK at the membrane upon stimulation. Through its interaction with IRAK, TRAF6 may undergo a conformational change, leading to its dissociation from IRAK, translocation to the cytosol, and formation of the TRAF6-TAK1-TAB1-TAB2 complex. Further modification of IRAK or TRAF6 might be required for the conformational change of the TRAF6-IRAK complex and the dissociation of TRAF6 from IRAK. We tested whether IL-1-induced IRAK degradation plays any role in the release of TRAF6 and TAB2 to the cytosol. We found that ALLM inhibited IRAK degradation, but it did not affect the dissociation of TRAF6 from IRAK nor the translocation of TRAF6 and TAB2 to the cytosol. Therefore, the IL-1-induced and IRAK-mediated translocation of TRAF6 and TAB2 probably does not occur through the degradation of IRAK. We have identified which domains of IRAK are required for the IL-1-induced translocation of TAB2 and TRAF6. The N-terminal (DD) and C-terminal domains (C1C2) of IRAK are required for both TAB2 and TRAF6 translocation. However, the N-proximal undetermined domain (UD) is required for TRAF6, but not TAB2, translocation (Fig. 3). These results suggest that IRAK mediates the translocation of TRAF6 and TAB2 separately. As proposed in our model (Fig. 10), IRAK might interact with TAB2 and TRAF6 simultaneously through its different domains to form a TAB2-IRAK-TRAF6 complex at the membrane, which would subsequently allow their independent disassociation from IRAK and separate translocation to the cytosol. Alternatively, IRAK might form distinct complexes with TAB2 and TRAF6, thereby mediating their translocation individually. TAK1 and TAB1 were localized primarily in the cytosol with or without stimulation, but small fractions of TAK1 and TAB1 were also detected in the membrane (Fig. 1 A). Although the TRAF6-TAK1-TAB1-TAB2 complex was detected in the cytosol (Fig. 4 B), we could not exclude the possibility that this complex first forms at the membrane with IRAK and then dissociates from IRAK and translocates to the cytosol. Previous studies (18Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1014) Google Scholar) have shown that the overexpression of TAK1 led to the activation of both NFκB and JNK. Furthermore, a dominant-negative mutant of TAK1 blocked the IL-1-induced activation of both NFκB and JNK (18Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1014) Google Scholar). Based on these studies, it was concluded that TAK1 is required for both IL-1-induced NFκB and JNK activation. We found that the domains of IRAK required for the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2 are also required for the IL-1-induced activation of NFκB (15, Fig. 9), indicating a tight correlation between the phosphorylation of these three molecules and NFκB activation. However, the deletion mutant dUD, which failed to activate NFκB and to support the IL-1-induced phosphorylation of TAK1, TAB1, and TAB2, still permitted IL-1-induced JNK activation in transfected I1A cells (15, Fig. 9), suggesting that the activation of TAK1, TAB1, and TAB2 is not necessary for the IL-1-induced activation of JNK. Supporting this conclusion, we have recently found (15Li X. Commane M. Jiang Z. Stark G.R. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4461-4465Crossref PubMed Scopus (143) Google Scholar) that the IL-1-induced phosphorylation of IRAK correlates strongly with the activation of NFκB but is not necessary for the activation of JNK. Therefore, the IL-1 signaling pathways leading to NFκB and JNK activation bifurcate at or upstream of IRAK and upstream of TAK1, TAB1, and TAB2. Our results suggest that the IRAK-mediated activation of TAK1, TAB1, and TAB2 is required for the IL-1-induced activation of NFκB but not of JNK. Analysis of tak1, tab1, and tab2 gene knock-out mice and cell lines derived from them will further elucidate the roles of these proteins in the IL-1 signaling pathway. We thank George Stark for insightful scientific discussions and careful editing of the draft manuscript; Zhaodan Cao for antibodies against IRAK and TRAF6 and critical scientific discussions; Tom Hamilton for helpful critiques on the text; Huiqin Nie for technical assistance; and Christine Kassuba for editorial support.}, number={45}, journal={Journal of Biological Chemistry}, author={Qian, Y. and Commane, M. and Ninomiya-Tsuji, J. and Matsumoto, K. and Li, X.}, year={2001}, pages={41661–41667} }
 @article{takaesu_ninomiya-tsuji_kishida_li_stark_matsumoto_2001, title={Interleukin-1 (IL-1) receptor-associated kinase leads to activation of TAK1 by inducing TAB2 translocation in the IL-1 signaling pathway}, volume={21}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035099717&partnerID=MN8TOARS}, DOI={10.1128/MCB.21.7.2475-2484.2001}, abstractNote={Interleukin-1 (IL-1) is a proinflammatory cytokine that recognizes a surface receptor complex and generates multiple cellular responses.IL-1 stimulation activates the mitogen-activated protein kinase kinase kinase TAK1, which in turn mediates activation of c-Jun N-terminal kinase and NF-B.TAB2 has previously been shown to interact with both TAK1 and TRAF6 and promote their association, thereby triggering subsequent IL-1 signaling events.The serine/threonine kinase IL-1 receptor-associated kinase (IRAK) also plays a role in IL-1 signaling, being recruited to the IL-1 receptor complex early in the signal cascade.In this report, we investigate the role of IRAK in the activation of TAK1.Genetic analysis reveals that IRAK is required for IL-1-induced activation of TAK1.We show that IL-1 stimulation induces the rapid but transient association of IRAK, TRAF6, TAB2, and TAK1.TAB2 is recruited to this complex following translocation from the membrane to the cytosol upon IL-1 stimulation.In IRAK-deficient cells, TAB2 translocation and its association with TRAF6 are abolished.These results suggest that IRAK regulates the redistribution of TAB2 upon IL-1 stimulation and facilitates the formation of a TRAF6-TAB2-TAK1 complex.Formation of this complex is an essential step in the activation of TAK1 in the IL-1 signaling pathway.}, number={7}, journal={Molecular and Cellular Biology}, author={Takaesu, G. and Ninomiya-Tsuji, J. and Kishida, S. and Li, X. and Stark, G.R. and Matsumoto, K.}, year={2001}, pages={2475–2484} }
 @article{hanada_ninomiya-tsuji_komaki_ohnishi_katsura_kanamaru_matsumoto_tamura_2001, title={Regulation of the TAK1 Signaling Pathway by Protein Phosphatase 2C}, volume={276}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035937111&partnerID=MN8TOARS}, DOI={10.1074/jbc.M007773200}, abstractNote={Protein phosphatase 2C (PP2C) is implicated in the negative regulation of stress-activated protein kinase cascades in yeast and mammalian cells. In this study, we determined the role of PP2Cβ-1, a major isoform of mammalian PP2C, in the TAK1 signaling pathway, a stress-activated protein kinase cascade that is activated by interleukin-1, transforming growth factor-β, or stress. Ectopic expression of PP2Cβ-1 inhibited the TAK1-mediated mitogen-activated protein kinase kinase 4-c-Jun amino-terminal kinase and mitogen-activated protein kinase kinase 6-p38 signaling pathways. In vitro, PP2Cβ-1 dephosphorylated and inactivated TAK1. Coimmunoprecipitation experiments indicated that PP2Cβ-1 associates with the central region of TAK1. A phosphatase-negative mutant of PP2Cβ-1, PP2Cβ-1 (R/G), acted as a dominant negative mutant, inhibiting dephosphorylation of TAK1 by wild-type PP2Cβ-1 in vitro. In addition, ectopic expression of PP2Cβ-1(R/G) enhanced interleukin-1-induced activation of an AP-1 reporter gene. Collectively, these results indicate that PP2Cβ negatively regulates the TAK1 signaling pathway by direct dephosphorylation of TAK1. Protein phosphatase 2C (PP2C) is implicated in the negative regulation of stress-activated protein kinase cascades in yeast and mammalian cells. In this study, we determined the role of PP2Cβ-1, a major isoform of mammalian PP2C, in the TAK1 signaling pathway, a stress-activated protein kinase cascade that is activated by interleukin-1, transforming growth factor-β, or stress. Ectopic expression of PP2Cβ-1 inhibited the TAK1-mediated mitogen-activated protein kinase kinase 4-c-Jun amino-terminal kinase and mitogen-activated protein kinase kinase 6-p38 signaling pathways. In vitro, PP2Cβ-1 dephosphorylated and inactivated TAK1. Coimmunoprecipitation experiments indicated that PP2Cβ-1 associates with the central region of TAK1. A phosphatase-negative mutant of PP2Cβ-1, PP2Cβ-1 (R/G), acted as a dominant negative mutant, inhibiting dephosphorylation of TAK1 by wild-type PP2Cβ-1 in vitro. In addition, ectopic expression of PP2Cβ-1(R/G) enhanced interleukin-1-induced activation of an AP-1 reporter gene. Collectively, these results indicate that PP2Cβ negatively regulates the TAK1 signaling pathway by direct dephosphorylation of TAK1. stress-activated protein kinase mitogen-activated protein kinase c-Jun amino-terminal kinase MAPK kinase MKK kinase protein serine/threonine phosphatase antibody hemagglutinin interleukin glutathioneS-transferase SDS-polyacrylamide gel electrophoresis mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase Stress-activated protein kinases (SAPKs)1 are a subfamily of the mitogen-activated protein kinase (MAPK) superfamily and are highly conserved from yeast to mammalian cells. SAPKs relay signals in response to various extracellular stimuli, including environmental stress and inflammatory cytokines. In mammalian cells, two distinct classes of SAPKs have been identified: the c-Jun amino-terminal kinases (JNKs) (JNK1, JNK2, and JNK3) and the p38 MAPKs (p38α, p38β, p38γ, and p38δ) (1Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1140) Google Scholar, 2Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1392) Google Scholar). Activation of SAPKs requires phosphorylation at conserved tyrosine and threonine residues in the catalytic domain. This phosphorylation is mediated by dual specificity protein kinases, which are the members of the MAPK kinase (MKK) family. Of these, MKK3 and MKK6 phosphorylate p38, MKK7 phosphorylates JNK, and MKK4 can phosphorylate either. These MKKs, in turn, are activated by phosphorylation of conserved serine and threonine residues (1Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1140) Google Scholar, 2Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1392) Google Scholar). Recently, several MKK-activating MKK kinases (MKKKs) have been identified. Some of these MKKKs are also known to be activated by phosphorylation, but the details are unclear at present. In the absence of signaling, SAPK cascades return to their inactive, dephosphorylated state, suggesting a possible role for phosphatases in SAPK regulation. In yeast cells, molecular genetic analysis has indicated that two distinct protein phosphatase groups, protein tyrosine phosphatase and protein serine/threonine phosphatase type 2C (PP2C), act as negative regulators of SAPK pathways (3Wurgler-Murphy S.M. Saito H. Trends. Biochem. Sci. 1997; 22: 172-176Abstract Full Text PDF PubMed Scopus (246) Google Scholar). InSchizosaccharomyces pombe, tyrosine phosphatase Pyp2 and the yeast homolog of PP2C (Ptc1 and Ptc3) have been shown to dephosphorylate and inactivate Spc1, the yeast homolog of SAPK (4Shiozaki K. Russell P. EMBO J. 1995; 14: 492-502Crossref PubMed Scopus (156) Google Scholar,5Nguyen A.N. Shiozaki K. Genes Dev. 1999; 13: 1653-1663Crossref PubMed Scopus (108) Google Scholar). PP2C is one of four major protein serine/threonine phosphatases (PP1, PP2A, PP2B, and PP2C) in eukaryotes and is implicated in the regulation of various cellular functions. To date, at least six distinct PP2C gene products (2Cα, 2Cβ, 2Cγ, 2Cδ, Wip1, and Ca2+/calmodulin-dependent protein kinase phosphatase) have been found in mammalian cells (6Tamura S. Lynch K.R. Larner J. Fox J. Yasui A. Kikuchi K. Suzuki Y. Tsuiki S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1796-1800Crossref PubMed Scopus (109) Google Scholar, 7Wenk J. Trompeter H.I. Pettrich K.G. Cohen P.T.W. Campbell D.G. Mieskes G. FEBS Lett. 1992; 297: 135-138Crossref PubMed Scopus (72) Google Scholar, 8Travis S.M. Welsh M.J. FEBS Lett. 1997; 412: 415-419Crossref PubMed Scopus (48) Google Scholar, 9Guthridge M.A. Bellosta P. Tavoloni N. Basilico C. Mol. Cell. Biol. 1997; 17: 5485-5498Crossref PubMed Scopus (53) Google Scholar, 10Tong Y. Quirion R. Shen S.-H. J. Biol. Chem. 1998; 273: 35282-35290Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 11Fiscella M. Zhang H. Fan S. Sakaguchi K. Shen S. Mercer W.E. Vande Woude G.F. O'Connor P.M. Appella E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6048-6053Crossref PubMed Scopus (470) Google Scholar, 12Kitani T. Ishida A. Okuno S. Takeuchi M. Kameshita I. Fujisawa H. J. Biochem. (Tokyo). 1999; 125: 1022-1028Crossref PubMed Scopus (51) Google Scholar). In addition, two distinct isoforms of the human PP2Cα (α-1 and -2) and five isoforms of the mouse PP2Cβ (β-1, -2, -3, -4, and -5) have been identified (13Mann D.J. Campbell D.G. McGowan C.H. Cohen P.T. Biochim. Biophys. Acta. 1992; 1130: 100-104Crossref PubMed Scopus (62) Google Scholar, 14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar, 15Terasawa T. Kobayashi T. Murakami T. Ohnishi M. Kato S. Tanaka O. Kondo H. Yamamoto H. Takeuchi T. Tamura S. Arch. Biochem. Biophys. 1993; 307: 342-349Crossref PubMed Scopus (42) Google Scholar, 16Kato S. Terasawa T. Kobayashi T. Ohnishi M. Sasahara Y. Kusuda K. Yanagawa Y. Hiraga A. Matsui Y. Tamura S. Arch. Biochem. Biophys. 1995; 318: 387-393Crossref PubMed Scopus (30) Google Scholar). These isoforms are generated in each species as splicing variants of a single pre-mRNA. We have recently reported that ectopic expression of mouse PP2Cα or PP2Cβ-1 inhibited the stress-activated MKK3/6-p38 and MKK4/7-JNK pathways but not the mitogen-activated MKK1-ERK1 pathway. Thus, negative regulation by PP2Cα and PP2Cβ-1 is selective for different SAPK pathways (17Hanada M. Kobayashi T. Ohnishi M. Ikeda S. Wang H. Katsura K. Yanagawa Y. Hiraga A. Kanamaru R. Tamura S. FEBS Lett. 1998; 437: 172-176Crossref PubMed Scopus (96) Google Scholar). Essentially the same results were obtained in studies of human PP2Cα-1 and -2 in mammalian cells (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar). Currently, the in vivo target molecule(s) of PP2C is unknown, although MKK4, MKK6, and p38 have been proposed as substrates of PP2Cα-2 (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar). TAK1 was originally identified as an MKKK that functions in the transforming growth factor-β signaling pathway (18Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1188) Google Scholar). TAK1 can activate both the MKK4-JNK and MKK6-p38 pathways (18Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1188) Google Scholar). Recent studies have indicated that TAK1 is also activated by various stimuli, including environmental stress and inflammatory cytokines, and that it plays critical roles in various cellular responses (19Shirakabe K. Yamaguchi K. Shibuya H. Irie K. Matsuda S. Moriguchi T. Gotoh Y. Matsumoto K. Nishida E. J. Biol. Chem. 1997; 272: 8141-8144Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 20Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (529) Google Scholar, 21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar, 22Ishitani T. Ninomiya-Tsuji J. Nagai S.-I. Nishita M. Meneghini M. Barker N. Waterman M. Bowerman B. Clevers H. Shibuya H. Matsumoto K. Nature. 1999; 399: 798-802Crossref PubMed Scopus (521) Google Scholar). Studies on the regulation of TAK1 activity have revealed that a TAK1-binding protein, TAB1, functions as an activator promoting TAK1 autophosphorylation (21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar, 23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). However, the protein phosphatase(s) responsible for inactivation of TAK1 has not been identified. In this study, we provide evidence indicating that PP2Cβ-1 selectively associates with TAK1 and inhibits the TAK1 signaling pathway by direct dephosphorylation. The restriction enzymes and other modifying enzymes used for DNA manipulation were obtained from Takara (Kyoto, Japan). Anti-6xHis, anti-Myc, and anti-TAK1 antibodies (Abs) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-MKK4 and anti-phospho-MKK3/6 Abs were supplied by New England Biolabs (Beverly, MA). Anti-hemagglutinin (HA; 12CA5) and anti-Flag (M2) Abs were purchased from Roche Molecular Biochemicals and Kodak Scientific Imaging Systems, respectively. Anti-PP2Cβ Ab was raised in rabbit against an oligopeptide of mouse PP2Cβ (RILSAENIPNLPPGGGLAGK). Human interleukin-1β (IL-1β) was from Roche Molecular Biochemicals. All the other reagents used were from Wako Pure Chemical (Osaka, Japan). Expression plasmids were constructed by standard procedures. Plasmids that express PP2C, TAK1, TAB1, MAPKs, MKKs, and MKKKs in mammalian cells were constructed using cDNAs encoding these proteins (17Hanada M. Kobayashi T. Ohnishi M. Ikeda S. Wang H. Katsura K. Yanagawa Y. Hiraga A. Kanamaru R. Tamura S. FEBS Lett. 1998; 437: 172-176Crossref PubMed Scopus (96) Google Scholar, 21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar) under the control of the CMV promoter. Epitope tags were added to the constructs using synthesized oligonucleotides. Mutated cDNAs were generated by polymerase chain reaction. For bacterial expression of proteins, cDNAs encoding the proteins were subcloned into pGEX (Amersham Pharmacia Biotech) to generate glutathioneS-transferase (GST) fusion proteins or into pQE31 (Qiagen, Hilden, Germany) to generate hexahistidine-tagged protein and affinity-purified by standard procedures. Other expression plasmids were as described elsewhere (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 24Kusuda K. Kobayashi T. Ikeda S. Ohnishi M. Chida N. Yanagawa Y. Shineha R. Nishihira T. Satomi S. Hiraga A. Tamura S. Biochem. J. 1998; 332: 243-250Crossref PubMed Scopus (42) Google Scholar) COS7, 293, and 293IL-1RI (25Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (781) Google Scholar) cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum. At 50–80% confluency the cells were transfected by the DEAE-dextran method or using LipofectAMINE (Life Technologies, Inc.). The total amount of DNA (0.5–2 μg per 35-mm dish) was kept constant by supplementing with empty vector. The cells were cultured for 24–48 h after transfection and then harvested. Immune complex kinase assays were performed as follows. The cells were lysed in a buffer containing 20 mm Tris-HCl, pH 7.5, 1% (v/v) Triton X-100, 150 mm NaCl, 1 mm EGTA, 1 mm sodium orthovanadate, 50 mm NaF, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride, and the lysates were incubated with appropriate Abs for 1 h at 4 °C. The resulting immune complexes were recovered with protein G-Sepharose (Amersham Pharmacia Biotech), washed twice with Tris-buffered saline (20 mm Tris-HCl, pH 7.5, 150 mm NaCl), twice with 20 mm Tris-HCl, pH 7.5, and then incubated with or without appropriate substrates in 25 μl of kinase buffer (20 mm Tris-HCl, pH 7.5, 10 mm MgCl2, and 1 mm dithiothreitol) containing 0.5–3 μCi of [γ-32P]ATP (NEG-002A, PerkinElmer Life Sciences) at 30 °C for 10–30 min. The reactions were stopped by adding SDS-sample buffer and boiled for 2 min. Protein phosphatase assays were carried out as follows. COS7 cells seeded onto 10-cm dishes were cotransfected with Flag-TAK1 and Myc-TAB1 expression plasmids. The Flag-TAK1-Myc-TAB1 complex was immunoprecipitated from cell extracts with anti-Flag Ab, and phosphorylation was carried out in kinase buffer containing [γ-32P]ATP at 30 °C for 30 min. After washing three times with 20 mm Tris-HCl, pH 7.5, the immune complex was then incubated with or without recombinant GST-PP2Cβ in kinase buffer at 30 °C for the indicated times. Phosphorylated proteins were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and the radioactivities incorporated into the proteins were detected with a BAS 2000 image analyzer (Fuji, Tokyo, Japan). Proteins in the cell lysates and immunoprecipitates were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The membranes were incubated with primary Abs at 4 °C for 16 h and then incubated with horseradish peroxidase-conjugated secondary Ab at 25 °C for 1 h. The chemiluminescence of each blot was detected with an enhanced chemiluminescence system (Amersham Pharmacia Biotech). Cells were lysed with a buffer containing 20 mm Tris-HCl, pH 7.5, 5 mm EDTA, 150 mm NaCl, 1% (v/v) Triton X-100, and 1 mmphenylmethylsulfonyl fluoride. The cell lysates were incubated with the indicated Abs for 1 h at 4 °C. The immunoprecipitated proteins were washed three times with Tris-buffered saline and submitted to Western blot analysis. Cells were transfected with the AP-1-luciferase reporter plasmid (26Beltman J. Erickson J.R. Martin G.A. Lyons J.F. Cook S.J. J. Biol. Chem. 1999; 274: 3772-3780Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). After the transfection, cells were treated with IL-1β for 6 h. Luciferase activity was determined with the Luciferase Assay System (Promega). β-actin-β-galactosidase reporter plasmid was cotransfected for normalizing transfection efficiencies. We have previously reported that two mouse PP2C isoforms, PP2Cα and PP2Cβ-1, selectively inhibit stress-activated MKKs (MKK3, MKK4, MKK6, and MKK7) (17Hanada M. Kobayashi T. Ohnishi M. Ikeda S. Wang H. Katsura K. Yanagawa Y. Hiraga A. Kanamaru R. Tamura S. FEBS Lett. 1998; 437: 172-176Crossref PubMed Scopus (96) Google Scholar). However, the target molecule(s) of PP2C has not been identified. Because both the MKK4-JNK and MKK6-p38 signaling pathways are activated by TAK1 (18Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1188) Google Scholar), we examined whether expression of PP2Cβ-1 affects TAK1-induced phosphorylation of MKK4 and MKK6 at their serine or threonine residues. Coexpression of TAK1 and TAB1 enhanced phosphorylation of MKK4 or MKK6 when expressed together in COS7 cells (Fig. 1, A and B). However, concomitant expression of PP2Cβ-1 markedly inhibited TAK1-induced phosphorylation of MKK4 and MKK6. We then tested whether PP2Cβ-1 expression affects TAK1-induced activation of JNK1 and p38α. Both the JNK1 and p38 kinases expressed in COS7 cells were activated by the exogenous TAK1. However, these kinase activities were inhibited when PP2Cβ-1 was coexpressed (Fig.1, C and D). In contrast, expression of PP2Cβ-1(R/G), a phosphatase-defective mutant containing an Arg-179 to Gly mutation, had no inhibitory effect on TAK1-induced activation of JNK1 or p38. These results suggest that PP2Cβ-1 inhibits the TAK1 signaling pathway at TAK1 or downstream of TAK1, e.g. MKKs and MAPKs. We have previously shown that TAK1, when coexpressed with TAB1, is activated by autophosphorylation (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). TAK1 autophosphorylation can be monitored by decreased mobility on SDS-PAGE, and this mobility shift was cancelled when Ser-192 of TAK1, which is the site of autophosphorylation, was mutated to alanine (Ref.23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar; also shown in Fig. 2). To determine whether PP2Cβ-1 affects the phosphorylation state of TAK1, we coexpressed TAK1, TAB1, and PP2Cβ-1 in COS7 cells. As shown in Fig. 2, expression of wild-type PP2Cβ-1, but not PP2Cβ-1(R/G), caused a substantial decrease in the levels of TAK1 phosphorylation. This result suggests that PP2Cβ-1 acts upon TAK1 directly. To investigate whether TAK1 is a substrate of PP2C, we examined the phosphorylation and kinase activity of TAK1 incubated with PP2Cin vitro. Flag-TAK1 and TAB1 were coexpressed in COS7 cells, and Flag-TAK1 was immunoprecipitated from cell extracts with anti-Flag Ab. When the immunopurified TAK1 complex was incubated with [γ-32P]ATP, TAK1 became autophosphorylated. This reaction mixture was next incubated with bacterially produced GST-PP2Cβ-1 or GST-PP2Cβ-1(R/G). TAK1 was found to be dephosphorylated by GST-PP2Cβ-1, but not by GST-PP2Cβ-1(R/G), in a dose-dependent manner (Fig.3 A). The PP2Cβ-1-mediated dephosphorylation reaction was dependent on the presence of Mg2+ (Fig. 3 B). We then determined whether dephosphorylation of TAK1 by PP2Cβ-1 reduces TAK1 activity. Flag-TAK1 immunoprecipitates were treated with GST-PP2Cβ-1 and measured for TAK1 activity in vitro. The presence of PP2Cβ-1 decreased the ability of TAK1 to phosphorylate itself and MKK6 (Fig. 3 C). Thus, PP2Cβ-1 dephosphorylates and inactivates TAK1 in vitro. This supports the possibility that PP2Cβ-1 negatively regulates the TAK1 signaling pathway by dephosphorylating TAK1. Recent studies have indicated that one of the human PP2C isoforms, PP2Cα-2, dephosphorylates and inactivates MKK4, MKK6, and p38 in vitro (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar). Therefore, we tested whether PP2Cβ-1 could also dephosphorylate and inactivate MKK6 in vitro. Bacterially produced MKK6 is activated by autophosphorylation and is able to phosphorylate p38 in vitro (27Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). We used this system to determine the effect of recombinant PP2Cβ-1 on MKK6 activity. We found that PP2Cβ-1 treatment did not affect MKK6 kinase activity under conditions where it inactivates TAK1 (Fig. 3, A andC versus Fig.4 A). Next, we determined the effect of PP2Cβ-1 on stress-induced phosphorylation of MKK6. COS7 cells were transfected with Flag-MKK6 and subjected to hyperosmotic stress, and Flag-MKK6 was immunoprecipitated from the cell lysates with anti-Flag Ab. The immunoprecipitates were then incubated with GST-PP2Cβ-1. Increasing concentrations of GST-PP2Cβ-1 had no effect on the phosphorylation level of MKK6 (Fig.4 B). Taken together, these results indicate that PP2Cβ-1 does not act upon MKK6. To determine whether PP2Cβ-1 associates with TAK1, we coexpressed Myc-TAK1 and HA-PP2Cβ-1 or HA-PP2Cβ-1(R/G) in COS7 cells. Cell extracts were immunoprecipitated with anti-Myc Ab, and coprecipitated HA-PP2Cβ-1 was detected by immunoblotting with anti-HA Ab. As shown in Fig.5 A, both HA-PP2Cβ-1 and HA-PP2Cβ-1(R/G) coimmunoprecipitated with Myc-TAK1, although the interaction of the wild-type PP2Cβ-1 with TAK1 was substantially weaker than that of PP2Cβ-1(R/G). This interaction is specific for PP2Cβ-1, because HA-PP2Cα, another major mouse PP2C isoform (28Kato S. Kobayashi T. Terasawa T. Ohnishi M. Sasahara Y. Kanamaru R. Tamura S. Gene. 1994; 145: 311-312Crossref PubMed Scopus (11) Google Scholar), did not coimmunoprecipitate with Myc-TAK1 under the same conditions (Fig. 5 B). Therefore, the association of PP2Cβ-1 with TAK1 is not caused by a nonspecific protein interaction. The observation that the catalytically inactive PP2Cβ has a higher affinity for TAK1 than that of wild-type PP2Cβ suggested that PP2Cβ might preferentially bind phosphorylated TAK1. The TAK1(S/A) mutant, in which Ser-192 is replaced by Ala, is defective in both phosphorylation and activation (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). We coexpressed PP2Cβ-1 and TAK1 or TAK1(S/A) in 293 cells and performed coimmunoprecipitation experiments. We found that TAK1(S/A) had an affinity for PP2Cβ-1 similar to that of wild-type TAK1 (Fig. 5 C), indicating that phosphorylation at Ser-192 is not required for association with PP2Cβ-1. We next examined whether endogenous PP2Cβ-1 and TAK1, expressed at lower physiological levels, can also interact with one another. As shown in Fig. 5 D, the 70-kDa endogenous TAK1 was specifically detected by anti-TAK1 Ab in the endogenous PP2Cβ immunoprecipitates from 293 cells, but not in the rabbit IgG immunoprecipitates. To determine which region of TAK1 is required for its interaction with PP2Cβ-1, we generated three Myc-tagged, truncated proteins, Myc-TAK1(N400), Myc-TAK1(C366), and Myc-TAK1(C176), containing the amino-terminal 400, carboxyl-terminal 366, and carboxyl-terminal 176 amino acids of TAK1, respectively (Fig.6 A). We coexpressed each deletion mutant along with Flag-PP2Cβ-1(R/G) in 293 cells and immunoprecipitated Flag-PP2Cβ-1(R/G) from cell extracts with anti-Flag Ab. Subsequent immunoblot analysis using anti-Myc Ab revealed that Flag-PP2Cβ-1 was associated with Myc-TAK1(N400) and Myc-TAK1(C366) but not with Myc-TAK1(C176) (Fig. 6 B). This result indicates that the central region of TAK1 is responsible for its association with PP2Cβ-1. To evaluate the specificity of the association of PP2Cβ-1 with TAK1, we examined whether PP2Cβ-1 could associate with other SAPK signaling pathway components. Flag-PP2Cβ-1 was coexpressed with Myc-TAK1, Myc-MEKK3, Myc-MKK4, Myc-MKK6, Myc-JNK1, or Myc-p38α in 293 cells. Flag-PP2Cβ-1 was immunoprecipitated from cell extracts with anti-Flag Ab, and the immune complexes were subjected to immunoblotting with anti-Myc Ab. None of these proteins, except for Myc-TAK1, coimmunoprecipitated with PP2Cβ-1 (Fig.7). Thus, PP2Cβ-1 specifically interacts with TAK1. Because PP2Cβ-1(R/G) appeared to have a higher affinity for TAK1 than did wild-type PP2Cβ-1 (Fig. 5 A), we asked whether PP2Cβ-1(R/G) could act as a dominant negative mutant. To test this possibility, we examined the effect of PP2Cβ-1(R/G) on PP2Cβ-1-mediated TAK1 dephosphorylation in vitro. We found that PP2Cβ-1(R/G) inhibited the dephosphorylation of TAK1 by PP2Cβ-1 in a dose-dependent manner (Fig.8 A). It has recently been reported that IL-1 treatment of cells activates the JNK signaling pathway through activation of TAK1 (21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar). Therefore, we examined the ability of PP2Cβ-1 to affect activation of TAK1 and AP-1 following IL-1 stimulation. We transfected 293IL-1RI cells with PP2Cβ-1 and TAK1 and determined the effect of PP2Cβ-1 expression on IL-1-induced mobility shift on SDS-PAGE and activation of TAK1. IL-1 treatment caused a slight mobility shift of TAK1 on SDS-PAGE, confirming our previous observation (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar) (Fig. 8 B,lower panel). However, the coexpression of PP2Cβ-1 totally reversed the mobility shift of TAK1. The expression of PP2Cβ-1 also inhibited the IL-1-induced activation of TAK1 (Fig. 8 B,upper panel). Next, we transfected 293IL-1RI cells with PP2Cβ-1 or/and PP2Cβ-1(R/G) and assayed AP-1 activity using an AP-1-dependent luciferase reporter gene. PP2Cβ-1 blocked IL-1-induced AP-1 activity in a dose-dependent manner (Fig.8 C). However, inhibition of IL-1-induced AP-1 activation by PP2Cβ-1 was reversed by cotransfection with PP2Cβ-1(R/G) (Fig.8 D). Furthermore, ectopic expression of PP2Cβ-1(R/G) enhanced IL-1-induced AP-1 activity in a dose-dependent manner (Fig. 8 E). MAPK cascades are intracellular signaling modules composed of three tiers of sequentially activating protein kinases: MKKK, MKK, and MAPK (1Garrington T.P. Johnson G.L. Curr. Opin. Cell Biol. 1999; 11: 211-218Crossref PubMed Scopus (1140) Google Scholar, 2Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1392) Google Scholar). Because phosphorylation of these components is essential for the activation of the MAPK cascades, protein phosphatases may be expected to play important roles in the regulation of these cascades. Indeed, we recently demonstrated that two major protein serine/threonine phosphatases, PP2Cα and PP2Cβ, inactivate the stress-activated JNK and p38 MAPK pathways (17Hanada M. Kobayashi T. Ohnishi M. Ikeda S. Wang H. Katsura K. Yanagawa Y. Hiraga A. Kanamaru R. Tamura S. FEBS Lett. 1998; 437: 172-176Crossref PubMed Scopus (96) Google Scholar). Furthermore, Takekawaet al. (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar) showed that PP2Cα inhibits the JNK and p38 cascades by dephosphorylating MKK4, MKK6, and p38. TAK1 is a member of the MKKK family and activates the JNK and p38 pathways. In this study we elucidated the role of PP2Cβ in TAK1-mediated signaling pathways. We present several lines of evidence suggesting that PP2Cβ negatively regulates the TAK1 pathways by dephosphorylating and inactivating TAK1. First, ectopic expression of PP2Cβ inhibits the MKK4-JNK and MKK6-p38 pathways activated by TAK1 (Fig. 1). Second, it is known that the TAK1-binding protein TAB1 activates TAK1 by promoting its autophosphorylation (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). We found that PP2Cβ overexpression decreased TAB1-induced TAK1 autophosphorylation in vivo(Fig. 2). Third, PP2Cβ dephosphorylates and inactivates TAK1 in vitro (Fig. 3) but fails to dephosphorylate MKK6 (Fig. 4). Finally, PP2Cβ interacts with TAK1 but not with MEKK3, MKK4, MKK6, JNK, or p38 (Figs. 5 and 7). Collectively, these data are consistent with the idea that PP2Cβ suppresses TAK1-mediated signaling by associating with and dephosphorylating TAK1. Because TAK1 functions in various biological responses, including acting as a positive regulator of transforming growth factor-β- and IL-1-induced signal transduction (18Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1188) Google Scholar, 21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar) and as a negative regulator in Wnt-induced signal transduction (22Ishitani T. Ninomiya-Tsuji J. Nagai S.-I. Nishita M. Meneghini M. Barker N. Waterman M. Bowerman B. Clevers H. Shibuya H. Matsumoto K. Nature. 1999; 399: 798-802Crossref PubMed Scopus (521) Google Scholar), it would be interesting to examine whether PP2Cβ contributes to the control of these physiological responses. Coexpression of TAK1/TAB1 with PP2Cβ-1 did not result in complete dephosphorylation of TAK1, as judged by the fact that the mobility of TAK1 is still slower than that of TAK1 expressed by itself (Fig. 2). COS7 cells contain a substantial amount of free, endogenous TAB1. Therefore, we speculate that the reason for the incomplete dephosphorylation may be that the dephosphorylated TAK1 can be rephosphorylated, because both TAB1 and ATP are present in the cells. Alternatively, this may suggest that there are other phosphorylation sites in TAK1 that are not substrates for PP2C. TAK1 associates with PP2Cβ but not with PP2Cα (Fig. 5). Thus, the interaction of TAK1 with PP2Cβ is rather specific. TAK1 is activated via autophosphorylation of Ser-192 in the activation loop between kinase domains VII and VIII. Mutation of TAK1 Ser-192 to Ala to create TAK1(S/A) abolishes both phosphorylation and activation of TAK1 (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). TAK1(S/A) has an affinity for PP2Cβ similar to that of wild-type TAK1 (Fig. 5 C), indicating that phosphorylation of TAK1 is not required for its association with PP2Cβ. This suggests that the association of TAK1 with PP2Cβ does not occur simply through affinity of the enzyme (PP2Cβ) for its substrate (phosphorylated TAK1), but rather that PP2Cβ and TAK1 are stably associated. This may ensure appropriate localization of PP2Cβ and facilitate the specific and rapid deactivation of TAK1. The central region of TAK1 is required for its association with PP2Cβ (Fig. 6). A similar region of TAK1 is involved in its association with TAB1 (20Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (529) Google Scholar), which suggests that PP2Cβ might prevent the association of TAK1 with TAB1. However, this possibility is unlikely, because we did not observe any competition between TAB1 and PP2Cβ in their association with TAK1. 2M. Hanada, J. Ninomiya-Tsuji, K.-i. Komaki, M. Ohnishi, K. Katsura, R. Kanamaru, K. Matsumoto, and S. Tamura, unpublished observation. Consistent with this, endogenous TAK1 constitutively associates with TAB1 in the absence of ligand stimulation (23Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). Therefore, the minimum regions of TAK1 required for association with PP2Cβ and TAB1 must be different. It is still not clear whether PP2Cβ associates with TAK1 directly or indirectly. However, the observation that PP2Cβ fails to interact with TAB12 argues against the possibility that TAB1 mediates the association between PP2Cβ and TAK1. To understand what role PP2C may play in regulating SAPK signaling pathways, it is important to determine how cellular PP2C activity is affected by extracellular stimuli. In fission yeast cells, the expression of Ptc1 is enhanced by hyperosmotic stress (29Gaits F. Shiozaki K. Russell P. J. Biol. Chem. 1997; 272: 17873-17879Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In contrast, expression levels of PP2Cα and PP2Cβ-1 are not altered following stress treatment of cells (17Hanada M. Kobayashi T. Ohnishi M. Ikeda S. Wang H. Katsura K. Yanagawa Y. Hiraga A. Kanamaru R. Tamura S. FEBS Lett. 1998; 437: 172-176Crossref PubMed Scopus (96) Google Scholar). PP2Cα has been shown to preferentially bind to the phosphorylated form of p38 and may function in the adaptive phase of the stimulation cycle to restore p38 to the inactive state following stimulation by stress (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar). PP2Cβ may play an analogous role in maintaining TAK1 signaling. TAK1 mediates IL-1-induced JNK signaling (21Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J.-I. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1032) Google Scholar), and ectopic expression of PP2Cβ blocks IL-1-induced AP-1 activation. PP2Cβ(R/G), a catalytically inactive mutant, has a higher affinity for TAK1 than does wild-type PP2Cβ and acts as a dominant negative factor, antagonizing the inhibitory effect of wild-type PP2Cβ on IL-1-induced AP-1 activation. Furthermore, ectopic expression of PP2Cβ(R/G) enhances IL-1-stimulated AP-1 activation but does not cause constitutive activation of AP-1.2 These results raise the possibility that PP2Cβ may down-regulate TAK1 activity after ligand stimulation. Because endogenous PP2Cβ constitutively associates with TAK1 (Fig. 5 D), and ligand stimulation does not affect this association,2 it is tempting to speculate that regulation of PP2Cβ enzymatic activity is involved in regulation of TAK1 signaling. Alternatively, PP2Cβ activity may be constitutive and serve to restore TAK1 to the inactive state following stimulation. Therefore it is important to determine whether the phosphatase activity of PP2Cβ is enhanced when cells are subjected to stress or treated with pro-inflammatory cytokines. Takekawa et al. (14Takekawa M. Maeda T. Saito H. EMBO J. 1998; 17: 4744-4752Crossref PubMed Scopus (244) Google Scholar) recently reported that PP2Cα dephosphorylates MKK4, MKK6, and p38 in vitro. In this study, we show that PP2Cβ dephosphorylates and inactivates TAK1. Thus, in mammalian cells, SAPK pathways are negatively regulated by multiple PP2C isoforms at different levels; PP2Cβ inhibits the pathways at the TAK1 MKKK level, and PP2Cα acts at the MKK and MAPK levels. In addition, two distinct groups of protein phosphatases other than PP2C also participate in the regulation of the SAPK pathways. The first group consists of the dual specificity phosphatases (also known as MAPK phosphatases) that inactivate MAPKs by dephosphorylating both tyrosine and threonine residues in the catalytic domains. Of the nine isolated MAPK phosphatases, M3/6 and MAPK phosphatase-5 have been shown to selectively dephosphorylate and inactivate p38 and JNK (30Muda M. Theodosius A. Rodrigues N. Boschert U. Camps M. Gillieron C. Davies K. Ashworth A. Arkinstall S. J. Biol. Chem. 1996; 271: 27205-27208Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 31Tanoue T. Moriguchi T. Nishida E. J. Biol. Chem. 1999; 274: 19949-19956Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The second group includes PP2A, which inactivates partially purified p38 kinase in vitro (32Rouse J. Cohen P. Trigon S. Morange M. Alonso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1523) Google Scholar). Cells treated with the PP2A inhibitor okadaic acid show enhanced MKK6 activity in epithelial cells (27Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). These results suggest that PP2A may also negatively regulate SAPK pathways and raise the possibility that several different groups of protein phosphatases each negatively regulate distinct targets in SAPK pathways. We are grateful to Drs. Ulrich Siebenlist (National Institutes of Health) and Simon J. Cook (the Babraham Institute) for providing us with the expression plasmids of MEKK3 and AP-1-luciferase, respectively. We are also grateful to Kimio Konno for technical assistance.}, number={8}, journal={Journal of Biological Chemistry}, author={Hanada, M. and Ninomiya-Tsuji, J. and Komaki, K.-I. and Ohnishi, M. and Katsura, K. and Kanamaru, R. and Matsumoto, K. and Tamura, S.}, year={2001}, pages={5753–5759} }
 @article{holtmann_enninga_kälble_thiefes_dörrie_broemer_winzen_wilhelm_ninomiya-tsuji_matsumoto_et al._2001, title={The MAPK Kinase Kinase TAK1 Plays a Central Role in Coupling the Interleukin-1 Receptor to Both Transcriptional and RNA-targeted Mechanisms of Gene Regulation}, volume={276}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0035793610&partnerID=MN8TOARS}, DOI={10.1074/jbc.M004376200}, abstractNote={Mechanisms of fulminant gene induction during an inflammatory response were investigated using expression of the chemoattractant cytokine interleukin-8 (IL-8) as a model. Recently we found that coordinate activation of NF-κB and c-Jun N-terminal protein kinase (JNK) is required for strong IL-8 transcription, whereas the p38 MAP kinase (MAPK) pathway stabilizes the IL-8 mRNA. It is unclear how these pathways are coupled to the receptor for IL-1, an important physiological inducer of IL-8. Expression of the MAP kinase kinase kinase (MAPKKK) TAK1 together with its coactivator TAB1 in HeLa cells activated all three pathways and was sufficient to induce IL-8 formation, NF-κB + JNK2-mediated transcription from a minimal IL-8 promoter, and p38 MAPK-mediated stabilization of a reporter mRNA containing IL-8-derived regulatory mRNA sequences. Expression of a kinase-inactive mutant of TAK1 largely blocked IL-1-induced transcription and mRNA stabilization, as well as formation of endogenous IL-8. Truncated TAB1, lacking the TAK1 binding domain, or a TAK1-derived peptide containing a TAK1 autoinhibitory domain were also efficient in inhibition. These data indicate that the previously described three-pathway model of IL-8 induction is operative in response to a physiological stimulus, IL-1, and that the MAPKKK TAK1 couples the IL-1 receptor to both transcriptional and RNA-targeted mechanisms mediated by the three pathways.}, number={5}, journal={Journal of Biological Chemistry}, author={Holtmann, H. and Enninga, J. and Kälble, S. and Thiefes, A. and Dörrie, A. and Broemer, M. and Winzen, R. and Wilhelm, A. and Ninomiya-Tsuji, J. and Matsumoto, K. and et al.}, year={2001}, pages={3508–3516} }
 @article{mochida_takeda_saitoh_nishitoh_amagasa_ninomiya-tsuji_matsumoto_ichijo_2000, title={ASK1 inhibits interleukin-1-induced NF-κB activity through disruption of TRAF6-TAK1 interaction}, volume={275}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0034693222&partnerID=MN8TOARS}, number={42}, journal={Journal of Biological Chemistry}, author={Mochida, Y. and Takeda, K. and Saitoh, M. and Nishitoh, H. and Amagasa, T. and Ninomiya-Tsuji, J. and Matsumoto, K. and Ichijo, H.}, year={2000}, pages={32747–32752} }
 @article{dan_watanabe_kobayashi_yamashita-suzuki_fukagaya_kajikawa_kimura_nakashima_matsumoto_ninomiya-tsuji_et al._2000, title={Molecular cloning of MINK, a novel member of mammalian GCK family kinases, which is up-regulated during postnatal mouse cerebral development}, volume={469}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0000827468&partnerID=MN8TOARS}, DOI={10.1016/S0014-5793(00)01247-3}, abstractNote={A new germinal center kinase (GCK) family kinase, Misshapen/NIKs-related kinase (MINK), has been cloned and its expression has been characterized in several tissues and various developmental stages of the mouse brain. MINK encodes a 1300 amino acid polypeptide, consisting of an N-terminal kinase domain, a proline-rich intermediate region, and a C-terminal GCK homology region. The expression of MINK is up-regulated during the postnatal development of the mouse brain. MINK activates the cJun N-terminal kinase and the p38 pathways.}, number={1}, journal={FEBS Letters}, author={Dan, I. and Watanabe, N.M. and Kobayashi, T. and Yamashita-Suzuki, K. and Fukagaya, Y. and Kajikawa, E. and Kimura, W.K. and Nakashima, T.M. and Matsumoto, K. and Ninomiya-Tsuji, J. and et al.}, year={2000}, pages={19–23} }
 @article{takaesu_kishida_hiyama_yamaguchi_shibuya_irie_ninomiya-tsuji_matsumoto_2000, title={TAB2, a novel adaptor protein, mediates activation of TAK1 MAPKKK by linking TAK1 to TRAF6 in the IL-1 signal transduction pathway}, volume={5}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033634977&partnerID=MN8TOARS}, number={4}, journal={Molecular Cell}, author={Takaesu, G. and Kishida, S. and Hiyama, A. and Yamaguchi, K. and Shibuya, H. and Irie, K. and Ninomiya-Tsuji, J. and Matsumoto, K.}, year={2000}, pages={649–658} }
 @article{kishimoto_matsumoto_ninomiya-tsuji_2000, title={TAK1 mitogen-activated protein kinase kinase kinase is activated by autophosphorylation within its activation loop}, volume={275}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0034629146&partnerID=MN8TOARS}, DOI={10.1074/jbc.275.10.7359}, abstractNote={TAK1, a member of the mitogen-activated kinase kinase kinase family, is activated <i>in vivo</i> by various cytokines, including interleukin-1 (IL-1), or when ectopically expressed together with the TAK1-binding protein TAB1. However, this molecular mechanism of activation is not yet understood. We show here that endogenous TAK1 is constitutively associated with TAB1 and phosphorylated following IL-1 stimulation. Furthermore, TAK1 is constitutively phosphorylated when ectopically overexpressed with TAB1. In both cases, dephosphorylation of TAK1 renders it inactive, but it can be reactivated by preincubation with ATP. A mutant of TAK1 that lacks kinase activity is not phosphorylated either following IL-1 treatment or when coexpressed with TAB1, indicating that TAK1 phosphorylation is due to autophosphorylation. Furthermore, mutation to alanine of a conserved serine residue (Ser-192) in the activation loop between kinase domains VII and VIII abolishes both phosphorylation and activation of TAK1. These results suggest that IL-1 and ectopic expression of TAB1 both activate TAK1 via autophosphorylation of Ser-192.}, number={10}, journal={Journal of Biological Chemistry}, author={Kishimoto, K. and Matsumoto, K. and Ninomiya-Tsuji, J.}, year={2000}, pages={7359–7364} }
 @article{kawasaki_hisamoto_lino_yamamoto_ninomiya-tsuji_matsumoto_1999, title={A Caenorhabditis elegans JNK signal transduction pathway regulates coordinated movement via type-D GABAergic motor neurons}, volume={18}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033168580&partnerID=MN8TOARS}, DOI={10.1093/emboj/18.13.3604}, number={13}, journal={EMBO Journal}, author={Kawasaki, M. and Hisamoto, N. and Lino, Y. and Yamamoto, M. and Ninomiya-Tsuji, J. and Matsumoto, K.}, year={1999}, pages={3604–3615} }
 @article{hanafusa_ninomiya-tsuji_masuyama_nishita_fujisawa_shibuya_matsumoto_nishida_1999, title={Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-β-induced gene expression}, volume={274}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033578721&partnerID=MN8TOARS}, DOI={10.1074/jbc.274.38.27161}, abstractNote={Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, is suggested to be involved in TGF-β-induced gene expression, but the signaling mechanism from TAK1 to the nucleus remains largely undefined. We have found that p38 mitogen-activated protein kinase, and its direct activator MKK6 are rapidly activated in response to TGF-β. Expression of dominant negative MKK6 or dominant negative TAK1 inhibited the TGF-β-induced transcriptional activation as well as the p38 activation. Constitutive activation of the p38 pathway in the absence of TGF-β induced the transcriptional activation, which was enhanced synergistically by coexpression of Smad2 and Smad4 and was inhibited by expression of the C-terminal truncated, dominant negative Smad4. Furthermore, we have found that activating transcription factor-2 (ATF-2), which is known as a nuclear target of p38, becomes phosphorylated in the N-terminal activation domain in response to TGF-β, that ATF-2 forms a complex with Smad4, and that the complex formation is enhanced by TGF-β. In addition, expression of a nonphosphorylatable form of ATF-2 inhibited the TGF-β-induced transcriptional activation. These results show that the p38 pathway is activated by TGF-β and is involved in the TGF-β-induced transcriptional activation by regulating the Smad-mediated pathway. Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, is suggested to be involved in TGF-β-induced gene expression, but the signaling mechanism from TAK1 to the nucleus remains largely undefined. We have found that p38 mitogen-activated protein kinase, and its direct activator MKK6 are rapidly activated in response to TGF-β. Expression of dominant negative MKK6 or dominant negative TAK1 inhibited the TGF-β-induced transcriptional activation as well as the p38 activation. Constitutive activation of the p38 pathway in the absence of TGF-β induced the transcriptional activation, which was enhanced synergistically by coexpression of Smad2 and Smad4 and was inhibited by expression of the C-terminal truncated, dominant negative Smad4. Furthermore, we have found that activating transcription factor-2 (ATF-2), which is known as a nuclear target of p38, becomes phosphorylated in the N-terminal activation domain in response to TGF-β, that ATF-2 forms a complex with Smad4, and that the complex formation is enhanced by TGF-β. In addition, expression of a nonphosphorylatable form of ATF-2 inhibited the TGF-β-induced transcriptional activation. These results show that the p38 pathway is activated by TGF-β and is involved in the TGF-β-induced transcriptional activation by regulating the Smad-mediated pathway. transforming growth factor-β mitogen-activated protein TGF-β-activated kinase 1 stress-activated protein kinase c-Jun N-terminal kinase activating transcription factor-2 TAK1-binding protein hemagglutinin TGF-β type I receptor TGF-β type II receptor mink lung epithelial. Members of the transforming growth factor-β (TGF-β)1 superfamily regulate cell proliferation, differentiation, and apoptosis. They exert their effects through heteromeric receptor complexes consisting of type I and type II serine/threonine kinase receptors. Following ligand binding, the type II receptor phosphorylates the type I receptor to activate it (1Massague J. Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 2Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3393) Google Scholar). Intracellular signaling downstream of these receptor complexes is mediated by the recently identified Smad family. To date, at least nine vertebrate Smad proteins have been identified (1Massague J. Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (829) Google Scholar, 2Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3393) Google Scholar, 3Derynck R. Feng X.H. Biochim. Biophys. Acta. 1997; 1333: 105-150Crossref PubMed Scopus (509) Google Scholar, 4Attisano L. Wrana J.L. Curr. Opin. Cell Biol. 1998; 10: 188-194Crossref PubMed Scopus (178) Google Scholar, 5Whitman M. Genes Dev. 1998; 12: 2445-2462Crossref PubMed Scopus (447) Google Scholar). Each member of the Smad family has different roles in signaling. For example, the receptor-regulated Smads, Smad1, -2, -3, -5, and -8, are phosphorylated within a conserved C-terminal SS(V/M)S motif by the specific type I receptors (6Eppert K. Scherer S.W. Ozcelik H. Pirone R. Hoodless P. Kim H. Tsui L.C. Bapat B. Gallinger S. Andrulis I.L. Thomsen G.H. Wrana J.L. Attisano L. Cell. 1996; 86: 543-552Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar, 7Hoodless P.A. Haerry T. Abdollah S. Stapleton M. O'Connor M.B. Attisano L. Wrana J.L. Cell. 1996; 85: 489-500Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar, 8Lechleider R.J. de Caestecker M.P. Dehejia A. Polymeropoulos M.H. Roberts A.B. J. Biol. Chem. 1996; 271: 17617-17620Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 9Abdollah S. Macias-Silva M. Tsukazaki T. Hayashi H. Attisano L. Wrana J.L. J. Biol. Chem. 1997; 272: 27678-27685Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 10Kretzschmar M. Liu F. Hata A. Doody J. Massague J. Genes Dev. 1997; 11: 984-995Crossref PubMed Scopus (479) Google Scholar, 11Liu X. Sun Y. Constantinescu S.N. Karam E. Weinberg R.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10669-10674Crossref PubMed Scopus (334) Google Scholar, 12Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (926) Google Scholar, 13Nakao A. Roijer E. Imamura T. Souchelnytskyi S. Stenman G. Heldin C.-H. ten Dijke P. J. Biol. Chem. 1997; 272: 2896-2900Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 14Souchelnytskyi S. Tamaki K. Engstrom U. Wernstedt C. ten Dijke P. Heldin C.-H. J. Biol. Chem. 1997; 272: 28107-28115Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar) and then associate with the common Smad, Smad4, which in turn translocates into the nucleus (6Eppert K. Scherer S.W. Ozcelik H. Pirone R. Hoodless P. Kim H. Tsui L.C. Bapat B. Gallinger S. Andrulis I.L. Thomsen G.H. Wrana J.L. Attisano L. Cell. 1996; 86: 543-552Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar,7Hoodless P.A. Haerry T. Abdollah S. Stapleton M. O'Connor M.B. Attisano L. Wrana J.L. Cell. 1996; 85: 489-500Abstract Full Text Full Text PDF PubMed Scopus (627) Google Scholar, 10Kretzschmar M. Liu F. Hata A. Doody J. Massague J. Genes Dev. 1997; 11: 984-995Crossref PubMed Scopus (479) Google Scholar, 12Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (926) Google Scholar, 15Lagna G. Hata A. Hemmati-Brivanlou A. Massague J. Nature. 1996; 383: 832-836Crossref PubMed Scopus (815) Google Scholar, 16Liu F. Hata A. Baker J.C. Doody J. Carcamo J. Harland R.M. Massague J. Nature. 1996; 381: 620-623Crossref PubMed Scopus (597) Google Scholar, 17Zhang Y. Feng X. We R. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (762) Google Scholar, 18Wu R.Y. Zhang Y. Feng X.H. Derynck R. Mol. Cell. Biol. 1997; 17: 2521-2528Crossref PubMed Scopus (186) Google Scholar, 19Zhang Y. Musci T. Derynck R. Curr. Biol. 1997; 7: 270-276Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 20Kawabata M. Inoue H. Hanyu A. Imamura T. Miyazono K. EMBO J. 1998; 17: 4056-4065Crossref PubMed Scopus (250) Google Scholar). Following nuclear translocation, Smads induce transcriptional activation of specific target genes through cooperation with other transcriptional factors. The Smad2·Smad4 complex interacts with the Mix.2 promoter through FAST1, a transcription factor that binds to an activin response element, and interacts with the goosecoid promoter through FAST2/FAST1 (21Chen X. Rubock M.J. Whitman M. Nature. 1996; 383: 691-696Crossref PubMed Scopus (635) Google Scholar, 22Labbe E. Silvestri C. Hoodless P.A. Wrana J.L. Attisano L. Mol. Cell. 1998; 2: 109-120Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 23Zhou S. Zawel L. Lengauer C. Kinzler K.W. Vogelstein B. Mol. Cell. 1998; 2: 121-127Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 24Liu F. Pouponnot C. Massague J. Genes Dev. 1997; 11: 3157-3167Crossref PubMed Scopus (402) Google Scholar). Similarly, at AP-1 binding sites in the promoter of TGF-β-responsive genes, the Smad3·Smad4 complex interacts with c-Jun/c-Fos (25Zhang Y. Feng X.H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (699) Google Scholar). Other transcription factors and coactivators such as TFE3, cAMP-response element-binding protein-binding protein/p300, and cAMP-response element-binding protein have been shown to interact with Smad complexes (25Zhang Y. Feng X.H. Derynck R. Nature. 1998; 394: 909-913Crossref PubMed Scopus (699) Google Scholar, 26Hua X. Liu X. Ansari D.O. Lodish H.F. Genes Dev. 1998; 12: 3084-3095Crossref PubMed Scopus (261) Google Scholar, 27Feng X.H. Zhang Y. Wu R.Y. Derynck R. Genes Dev. 1998; 12: 2153-2163Crossref PubMed Scopus (457) Google Scholar, 28Janknecht R. Wells N.J. Hunter T. Genes Dev. 1998; 12: 2114-2119Crossref PubMed Scopus (441) Google Scholar, 29Pouponnot C. Jayaraman L. Massague J. J. Biol. Chem. 1998; 273: 22865-22868Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 30Topper J.N. DiChiara M.R. Brown J.D. Williams A.J. Falb D. Collins T. Gimbrone Jr., M.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9506-9511Crossref PubMed Scopus (155) Google Scholar, 31Szuts D. Eresh S. Bienz M. Genes Dev. 1998; 12: 2022-2035Crossref PubMed Scopus (89) Google Scholar). Thus, the mechanism of transcriptional activation by Smads is likely, at least partly, based on the interaction with other transcription factors. Although previous studies indicated that TAK1, a member of the MAP kinase kinase kinase family, is involved in the TGF-β signaling pathway (32Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1188) Google Scholar, 33Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (529) Google Scholar), the signaling mechanism to the nucleus remains largely undefined. TAK1 is a potent activator of the p38 pathway and the SAPK/JNK pathway (34Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 35Shirakabe K. Yamaguchi K. Shibuya H. Irie K. Matsuda S. Moriguchi T. Gotoh Y. Matsumoto K. Nishida E. J. Biol. Chem. 1997; 272: 8141-8144Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 36Wang W. Zhou G. Hu M.C.-T. Yao Z. Tan T.H. J. Biol. Chem. 1997; 272: 22771-22775Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). However, it is not known whether TAK1 actually acts as a physiological activator of these signaling pathways in vivo. Recently, it was reported that the SAPK/JNK pathway is required for TGF-β-mediated signaling (36Wang W. Zhou G. Hu M.C.-T. Yao Z. Tan T.H. J. Biol. Chem. 1997; 272: 22771-22775Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 37Atfi A. Buisine M. Mazars A. Gespach C. J. Biol. Chem. 1997; 272: 24731-24734Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 38Atfi A. Djelloul S. Chastre E. Davis R. Gespach C. J. Biol. Chem. 1997; 272: 1429-1432Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). But the activation of SAPK/JNK by TGF-β is maximal 12 h after stimulation, whereas TGF-β induces the activation of TAK1 within 15 min. These results seemed to imply that TAK1 is not a direct activator of the SAPK/JNK pathway in TGF-β signaling. Thus we have investigated which pathway plays a pivotal role downstream of TAK1 in TGF-β signaling. Furthermore, we have examined the possible functional link between the Smad and TAK1 pathways in TGF-β signaling. Here we report that the MKK6-p38 kinase cascade appears to lie downstream of TAK1 in TGF-β signaling, and the transcription factor ATF-2 functions as one of targets of this pathway. ATF-2 can associate with Smad4 in response to TGF-β. Our results suggest that TGF-β activates the Smad and TAK1 pathways, resulting in the formation of an active transcription complex containing Smad4 and ATF-2. For assaying endogenous p38, SAPK/JNK, MKK6, and ATF-2 activities, 2 × 105 cells were lysed in 150 μl of lysis buffer containing 20 mm Tris-HCl (pH 7.5), 12.5 mm2-glycerophosphate, 150 mm NaCl, 1.5 mmMgCl2, 2 mm EGTA, 10 mm NaF, 0.5% Triton X-100, 2 mm dithiothreitol, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, and 20 μg/ml aprotinin. Cell lysates (20 μl/lane) were subjected to immunoblotting with the indicated anti-phospho-specific antibodies. For anti-Myc immunoprecipitations, cell lysates were incubated with anti-Myc 9E10 antibody and protein G-Sepharose beads (Amersham Pharmacia Biotech) for 2 h at 4 °C with rocking. The beads were washed three times with ice-cold phosphate-buffered saline and subjected to kinase assays or association assays. C2C12 cells were cultured in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum. mink lung epithelial (Mv1Lu) cells were cultured in Dulbecco's modified Eagle's medium/F-12 containing 5% fetal bovine serum. These cells were transfected using LipofectAMINE according to the manufacturer's instructions (Life Technologies, Inc.). For protein kinase assays, we prepared cell lysates from 5 × 105cells that were transiently transfected with the indicated constructs (∼20–50% transfection efficiencies). Immunocomplex kinase reactions of Myc-MKK6 and Myc-p38 were performed in a final volume of 15 μl containing 20 mm Tris-HCl (pH 7.5), 2 mm EGTA, 15 mm MgCl2, 100 μm[γ-32P]ATP, and 3 μg of His-tagged kinase-negative MPK-2 or glutathione S-transferase-tagged ATF-2. Samples were incubated at 30 °C for 20 min. Reactions were terminated by the addition of sample buffer and boiling. Substrate phosphorylations were detected and quantified by autoradiography and image analysis (Bio-Rad). For luciferase reporter assays, cells were transiently transfected with p3TP-Lux, which contains TGF-β-responsive elements (40Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1437) Google Scholar), pCMVβ-gal, and the indicated constructs or with empty vector alone. The total amount of DNA for each transfection was kept constant using empty vector. Cells were treated for 15–20 h with or without 5–10 ng/ml TGF-β, and luciferase activity in cell lysates was measured using the luciferase assay system (Promega) in a Berthold Lumat LB 9507 luminometer. To determine transfection efficiency in each assay, β-galactosidase activity was measured according to the protocol of Promega, and the data were normalized for β-galactosidase activity. C2C12 cells cotransfected with hemagglutinin (HA)-tagged XSmad2, Myc-tagged XSmad4, and the indicated plasmids were treated with TGF-β for 60 min and then were lysed in TNE buffer containing 10 mm Tris-HCl (pH 7.8), 150 mm NaCl, 1 mm EDTA, 1% (v/v) Nonidet P-40, 2 mm dithiothreitol, 1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, and 20 μg/ml aprotinin. The total amount of DNA for each transfection was kept constant using empty vector. Cell lysates were subjected to anti-Myc immunoprecipitation as described above. XSmad complexes in the immunoprecipitates and in total lysates were separated by SDS-polyacrylamide gel electrophoresis and detected by immunoblotting as indicated. Recent studies have shown that TAK1 can activate the p38 and SAPK/JNK pathways in vitro and when overexpressed in cells (34Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 35Shirakabe K. Yamaguchi K. Shibuya H. Irie K. Matsuda S. Moriguchi T. Gotoh Y. Matsumoto K. Nishida E. J. Biol. Chem. 1997; 272: 8141-8144Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 36Wang W. Zhou G. Hu M.C.-T. Yao Z. Tan T.H. J. Biol. Chem. 1997; 272: 22771-22775Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar), but the physiological significance of this reaction is unclear. Because TAK1 has been shown to function as a mediator of the TGF-β-induced transcriptional activation (32Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1188) Google Scholar), we investigated whether p38 or SAPK/JNK also functions in TGF-β signaling. To determine whether p38 or SAPK/JNK is activated in response to TGF-β, we first tested these kinase activities using antibodies specific for the phosphorylated forms of p38 or SAPK/JNK, respectively. The immunoblotting of whole cell extracts showed that slight activation of p38 was detected within 15 min of TGF-β stimulation, and marked activation was observed at 30–60 min in C2C12 cells (Fig.1 A, TGF-β, αP-p38). The level of the activation was about one-fourth or one-fifth of the maximal activation that was attained by osmotic shock (Fig. 1 A, NaCl). The total amount of p38 was unchanged during stimulation (Fig. 1 A, αp38). In contrast to p38, there was no apparent activation of SAPK/JNK within 60 min of TGF-β stimulation (Fig.1 A, αP-SAPK/JNK). Essentially identical results were obtained in Mv1Lu cells and HaCaT cells (data not shown). Then we focused on the p38 pathway. We have previously demonstrated that MKK6, a member of the MAP kinase kinase family, can act as a strong activator of p38 (34Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 39Moriguchi T. Toyoshima F. Gotoh Y. Iwamatsu A. Irie K. Mori E. Kuroyanagi N. Hagiwara M. Matsumoto K. Nishida E. J. Biol. Chem. 1996; 271: 26981-26988Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). We tested whether MKK6 is also activated by TGF-β in C2C12 cells. The anti-phospho immunoblotting of whole cell extracts showed that activation of MKK6 was observed within 15 min of TGF-β stimulation and peaked at 45 min (Fig. 1 B). The slight activation of MKK3, another activator of p38, was also observed (Fig.1 B). The total amounts of MKK6 and MKK3 did not change during stimulation (Fig. 1 B). Next, Myc epitope-tagged MKK6 or p38 was transiently transfected into C2C12 cells. After treatment of the cells with TGF-β, their activities were determined by in vitro kinase assays using kinase-negative MPK-2 and ATF-2, respectively, as substrates. Both the transfected MKK6 and the transfected p38 were activated in response to TGF-β, with the time courses of their activation being the same as those of endogenous MKK6 and p38 (Fig. 1 C, cf. Fig. 1, A and B). The TGF-β-induced activation of MKK6 was blocked by expression of dominant negative TAK1, TAK1(K63W) (Fig. 1 D, upper left panel). The TGF-β-induced activation of p38 was also blocked by expression of TAK1(K63W). It was inhibited by expression of dominant negative TGF-β type I receptor, TβRI(K232R) or by that of dominant negative MKK6, MKK6(AA) (Fig. 1 D, upper right panel). The anti-phospho immunoblotting of whole cell extracts showed that the activation of endogenous p38 by TGF-β was also inhibited by expression of TβRI(K232R), TAK1(K63W), or MKK6(AA) (Fig. 1 D, lower panel). In these experiments, transfection efficiencies of dominant negative constructs were ∼50% and endogenous p38 in whole cells was assayed, so it is reasonable that only partial inhibitions were seen, and about 50% inhibitions seen might mean almost complete inhibition. These results demonstrate that the MKK6-p38 cascade is activated in response to TGF-β through TAK1. To test possible involvement of the p38 pathway in the induction of gene expression by TGF-β, we examined the effect of dominant negative mutants of TAK1 and MKK6 on TGF-β-induced transcriptional activation. We used the p3TP-Lux reporter construct containing a luciferase gene controlled by a TGF-β-inducible promoter (40Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1437) Google Scholar). Transient transfection of p3TP-Lux into Mv1Lu cells resulted in a strong induction of luciferase activity in response to TGF-β (Fig.2 A). Cotransfection of an expression plasmid encoding TAK1(K63W) or MKK6(AA) inhibited activation of 3TP promoter by TGF-β in Mv1Lu cells (Fig. 2 A). Similar results were obtained with C2C12 cells, and the inhibitory effect of MKK6(AA) on transcriptional activation by TGF-β was dose-dependent (Fig. 2 A). Cotransfection of a plasmid encoding CL100, which is a dual-specificity phosphatase acting on members of the MAP kinase superfamily including p38, also resulted in inhibition of the transcriptional activation by TGF-β (data not shown). Moreover, expression of a constitutively active MKK6, MKK6(DE), induced the transcriptional activation in the absence of TGF-β, and coexpression of p38 enhanced this transcriptional activation (Fig.2 B). These results indicate that a kinase cascade consisting of TAK1, MKK6, and p38 is involved in the induction of gene expression by TGF-β. Because it has been shown that Smad proteins play an essential role in TGF-β signaling, we examined the relationship between the Smad and p38 pathways. As previously reported (12Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (926) Google Scholar, 16Liu F. Hata A. Baker J.C. Doody J. Carcamo J. Harland R.M. Massague J. Nature. 1996; 381: 620-623Crossref PubMed Scopus (597) Google Scholar, 17Zhang Y. Feng X. We R. Derynck R. Nature. 1996; 383: 168-172Crossref PubMed Scopus (762) Google Scholar), overexpression of Smad2 and Smad4 induced activation of the 3TP promoter in the absence of TGF-β. Although expression of either TAK1(WT) or MKK6(DE) induced some modest increase of transcriptional activation, coexpression with Smad2 and Smad4 resulted in a strong induction of 3TP promoter activation (Fig. 3). Coexpression of Smad2 and Smad4 along with TAK1 and TAB1, which is an activator of TAK1, resulted in further synergetic activation of 3TP promoter, and this activation was enhanced by expression of MKK6 (data not shown). As shown in Fig. 4 A, the MKK6(DE)-induced transcriptional activation was effectively inhibited by the C-terminal truncated type of Smad4 (Smad4ΔC), which is known as the dominant interfering type of Smad4. On the other hand, TAK1(K63W) and MKK6(AA) scarcely inhibited the transcriptional activation induced by overexpression of Smad2 and Smad4 (Fig.4 B). In a control experiment, coexpression of Smad4ΔC completely abolished the Smad2/4-induced transcriptional activation (Fig. 4 B). These results suggest that the TAK1-MKK6-p38 pathway interacts cooperatively with the Smad pathway to mediate signaling of TGF-β to the nucleus and that the signaling of the p38 pathway requires the Smad pathway.Figure 4Relationship between the TAK1-MKK6-p38 pathway and Smad proteins in TGF-β signaling. A, effect of the C-terminal truncated type (ΔC) of Smad4 on the activated MKK6(DE)-induced reporter activity is shown. C2C12 cells were transfected with p3TP-Lux (0.2 μg) together with plasmids encoding MKK6(DE) (0.3 μg) and Smad4ΔC (0.3 μg) or an empty vector plasmid. After 24 h, cells were harvested and assayed for luciferase activity. The relative luciferase activities (means ± S.D.; n = 3) are presented. The experiment was repeated three times with similar results. B, effect of dominant negative TAK1 or dominant negative MKK6 on Smad-induced reporter activity is shown. C2C12 cells were transfected with p3TP-Lux (0.2 μg) and the indicated combinations of Smad2 and Smad4 (Smad2/4), Smad4ΔC, TAK1(K63W), and MKK6(AA). After 24 h, cells were harvested and assayed for luciferase activity. The relative luciferase activities (means ± S.D.; n = 3) are presented. The experiment was repeated three times with similar results. C, effect of the TAK1-MKK6-p38 pathway on TGF-β-induced interaction of Smad2 with Smad4 is shown. C2C12 cells were transfected with SRα-HA-Xenopus Smad2 (a gift from Dr. D. A. Melton) and SRα-Myc-Xenopus Smad4 together with TβRI(K232R) (TβR1(KR)), TAK1(K63W), or MKK6(AA) or the activated forms of TAK1(ΔN) (ΔNTAK1) as indicated. Cell lysates were subjected to immunoprecipitation (IP) with anti-Myc 9E10 antibody and then immunoblotted (IB) using anti-HA Y11 antibody or anti-Myc 9E10 antibody. To confirm equivalent levels of Smad2 expression, aliquots of total lysates were immunoblotted with anti-HA antibody. The experiment was repeated three times with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To investigate the possibility that p38 might regulate Smad proteins directly, we tested the effect of the TAK1-MKK6-p38 pathway on association of Smad2 and Smad4 in response to TGF-β. Coexpression of Smad2 and Smad4 in C2C12 cells resulted in formation of a heteromeric complex in a TGF-β stimulation-dependent manner (Fig.4 C). This association was significantly decreased in the presence of TβRI(KR), the dominant interfering TβRI mutant. In contrast, coexpression of TAK1(K63W) or MKK6(AA) did not affect the association (Fig. 4 C). A constitutively active TAK1, ΔNTAK1, did not affect it either (Fig. 4 C). These results suggest that the TAK1-MKK6-p38 pathway does not directly regulate the association of Smads. As the other likely mechanism by which the TAK1-MKK6-p38 pathway regulates the TGF-β-induced transcriptional activation, we then hypothesized the possibility of p38-mediated phosphorylation of transcription factors. The 3TP promoter contains three consecutive 12-O-tetradecanoylphorbol-13-acetate response elements and a portion of the plasminogen activator inhibitor 1 promoter region that contains putative AP-1 sites (40Wrana J.L. Attisano L. Carcamo J. Zentella A. Doody J. Laiho M. Wang X.F. Massague J. Cell. 1992; 71: 1003-1014Abstract Full Text PDF PubMed Scopus (1437) Google Scholar). Among the transcription factors known to bind to the AP-1 element, ATF-2 has been shown to be phosphorylated by p38 on Thr69 and Thr71(41Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (481) Google Scholar, 42Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1352) Google Scholar, 43van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (573) Google Scholar). Phosphorylation of ATF-2 on these sites causes an increase in transcriptional activity in vivo (41Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (481) Google Scholar, 42Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1352) Google Scholar, 43van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (573) Google Scholar). To determine whether TGF-β stimulates phosphorylation of ATF-2 at these threonine residues, we analyzed phosphorylation at these sites using an antibody specific for the Thr71-phosphorylated form of ATF-2. Little, if any, phospho-ATF-2 was detected in Mv1Lu cells in the absence of TGF-β signaling, whereas TGF-β treatment stimulated the phosphorylation of endogenous ATF-2 on Thr71 (Fig.5 A). We next analyzed the effect of the TAK1 pathway on the phosphorylation of ATF-2 in human embryonic kidney epithelial 293 cells, which lack any detectable expression of the endogenous TGF-β type II receptor (TβRII) (44Ebner R. Chen R.H. Shum L. Lawler S. Zioncheck T.F. Lee A. Lopez A.R. Derynck R. Science. 1993; 260: 1344-1348Crossref PubMed Scopus (368) Google Scholar). When 293 cells were transiently transfected with HA epitope-tagged ATF-2, TβRI, and TβRII and treated with TGF-β, we observed TGF-β-stimulated phosphorylation of ATF-2 (Fig. 5 B, left). Some phosphorylation of ATF-2 in the absence of ligand is also observed and is likely caused by overexpression of TβRI and TβRII in 293 cells, which drives their ligand-independent association and consequent activation of TβRI. Activation of TAK1 by cotransfection of TAB1 and TAK1 caused enhanced phosphorylation of ATF-2 (Fig. 5 B, right). These results suggest that ATF-2 phosphorylation in response to TGF-β signaling is mediated by the TAK1 pathway. This is consistent with the observation that TAK1 regulates the MKK6-p38 cascade. Because ATF-2 is localized in the nucleus, it is likely that TGF-β stimulation induces the nuclear accumulation of Smads and consequent association with ATF-2. To examine this possibility, we tested the interaction between ATF-2 and Smad4. Coexpression of ATF-2 and Smad4 in COS7 cells resulted in formation of a complex, and this association was enhanced in response to TGF-β (Fig. 5 C). Furthermore, overexpression of ATF-2(Ala69/Ala71), a nonphosphorylated form of ATF-2 in which Thr69 and Thr71 are replaced by alanine residues, inhibited transcriptional activation induced by TGF-β (Fig. 5 D). Taken together, these observations suggest that Smad complexes and phosphorylated ATF-2 participate in a complex that binds to DNA sequences present in the region of p3TP-Lux, resulting in its transcriptional activation. The present and previous studies have demonstrated that the TGF-β signal activates two independent pathways, the TAK1-mediated and the Smad-mediated pathways (2Heldin C.-H. Miyazono K. ten Dijke P. Nature. 1997; 390: 465-471Crossref PubMed Scopus (3393) Google Scholar, 32Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1188) Google Scholar, 34Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 35Shirakabe K. Yamaguchi K. Shibuya H. Irie K. Matsuda S. Moriguchi T. Gotoh Y. Matsumoto K. Nishida E. J. Biol. Chem. 1997; 272: 8141-8144Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 45Shibuya H. Iwata H. Masuyama N. Gotoh Y. Yamaguchi K. Irie K. Matsumoto K. Nishida E. Ueno N. EMBO J. 1998; 17: 1019-1028Crossref PubMed Scopus (192) Google Scholar). In the Smad pathway, TGF-β stimulation leads to the direct phosphorylation of Smad2 and Smad3 by the type I receptor kinase, consequent hetero-oligomer formation with Smad4, and accumulation in the nucleus (9Abdollah S. Macias-Silva M. Tsukazaki T. Hayashi H. Attisano L. Wrana J.L. J. Biol. Chem. 1997; 272: 27678-27685Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 11Liu X. Sun Y. Constantinescu S.N. Karam E. Weinberg R.A. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10669-10674Crossref PubMed Scopus (334) Google Scholar, 12Nakao A. Imamura T. Souchelnytskyi S. Kawabata M. Ishisaki A. Oeda E. Tamaki K. Hanai J. Heldin C.-H. Miyazono K. ten Dijke P. EMBO J. 1997; 16: 5353-5362Crossref PubMed Scopus (926) Google Scholar, 13Nakao A. Roijer E. Imamura T. Souchelnytskyi S. Stenman G. Heldin C.-H. ten Dijke P. J. Biol. Chem. 1997; 272: 2896-2900Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 46Macias-Silva M. Abdollah S. Hoodless P.A. Pirone R. Attisano L. Wrana J.L. Cell. 1996; 87: 1215-1224Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar,47Souchelnytskyi S. ten Dijke P. Miyazono K. Heldin C.-H. EMBO J. 1996; 15: 6231-6240Crossref PubMed Scopus (109) Google Scholar). Our results indicate that in the TAK1 pathway TGF-β activates the TAK1-MKK6-p38 kinase cascade leading to the phosphorylation of ATF-2, and ATF-2 associates with Smad4 in response to TGF-β. Therefore, Smad complexes and phosphorylated ATF-2 may interact in a nucleoprotein complex that associates with DNA and activates transcription of TGF-β-responsive genes. Two papers reporting a similar conclusion appeared after submission of this article (48Sano Y. Harada J. Tashiro S. Gotoh-Mandeville R. Maekawa T. Ishii S. J. Biol. Chem. 1999; 274: 8949-8957Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 49Adachi-Yamada T. Nakamura M. Irie K. Tomoyasu Y. Sano Y. Mori E. Gotoh S. Ueno N. Nishida Y. Matsumoto K. Mol. Cell. Biol. 1999; 19: 2322-2329Crossref PubMed Google Scholar). In our study, dominant negative Smad4 could inhibit the p38 pathway-dependent transcriptional activation efficiently, although dominant negative TAK1 or dominant negative MKK6 could not inhibit the Smad2·Smad4-induced transcriptional activation. Thus, the Smad pathway is essential. It is likely that the affinity of Smad2 and Smad4 for DNA in the absence of TGF-β is low or insufficient, and other transcription factors such as ATF-2 may enhance or stabilize Smad·DNA complexes in a ligand-dependent manner. Overexpression of Smad2 and Smad4 may be sufficient for efficient binding to DNA even in the absence of TGF-β to activate transcription of target genes. On the other hand, expression of a nonphosphorylated form of ATF-2 could inhibit the TGF-β-induced transcriptional activation completely, whereas dominant negative MKK6 could inhibit it significantly but not completely. It is possible that other MAP kinase-related pathways such as JNK/SAPK and classical MAP kinase pathways are involved in the transcriptional activation through phosphorylation of ATF-2 or ATF-2-related transcriptional factors. We thank R. Derynck, S. Ishii, J. Massague, D. A. Melton, K. Miyazono, and J. L. Wrana for materials.}, number={38}, journal={Journal of Biological Chemistry}, author={Hanafusa, H. and Ninomiya-Tsuji, J. and Masuyama, N. and Nishita, M. and Fujisawa, J.-I. and Shibuya, H. and Matsumoto, K. and Nishida, E.}, year={1999}, pages={27161–27167} }
 @article{meneghini_ishitani_carter_hisamoto_ninomiya-tsuji_thorpe_hamill_matsumoto_bowerman_1999, title={Map kinase and Wnt pathways converge to downregulate an HMG-domain repressor in Caenorhabditis elegans}, volume={399}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033600227&partnerID=MN8TOARS}, DOI={10.1038/21666}, number={6738}, journal={Nature}, author={Meneghini, M.D. and Ishitani, T. and Carter, J.C. and Hisamoto, N. and Ninomiya-Tsuji, J. and Thorpe, C.J. and Hamill, D.R. and Matsumoto, K. and Bowerman, B.}, year={1999}, pages={793–797} }
 @article{ishitani_ninomiya-tsuji_nagai_nishita_meneghini_barker_waterman_bowerman_clevers_shibuya_et al._1999, title={The TAK1-NLK-MAPK-related pathway antagonizes signalling between β- catenin and transcription factor TCF}, volume={399}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033600239&partnerID=MN8TOARS}, DOI={10.1038/21674}, number={6738}, journal={Nature}, author={Ishitani, T. and Ninomiya-Tsuji, J. and Nagai, S.-I. and Nishita, M. and Meneghini, M. and Barker, N. and Waterman, M. and Bowerman, B. and Clevers, H. and Shibuya, H. and et al.}, year={1999}, pages={798–802} }
 @article{ninomiya-tsuji_kishimoto_hiyama_inoue_cao_matsumoto_1999, title={The kinase TAK1 can activate the NIK-IκB as well as the MAP kinase cascade in the IL-1 signalling pathway}, volume={398}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033580466&partnerID=MN8TOARS}, DOI={10.1038/18465}, number={6724}, journal={Nature}, author={Ninomiya-Tsuji, J. and Kishimoto, K. and Hiyama, A. and Inoue, J.-I. and Cao, Z. and Matsumoto, K.}, year={1999}, pages={252–256} }
 @article{yamaguchi_nagai_ninomiya-tsuji_nishita_tamai_irie_ueno_nishida_shibuya_matsumoto_1999, title={XIAP, a cellular member of the inhibitor of apoptosis protein family, links the receptors to TAB1-TAK1 in the BMP signaling pathway}, volume={18}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0033521529&partnerID=MN8TOARS}, DOI={10.1093/emboj/18.1.179}, number={1}, journal={EMBO Journal}, author={Yamaguchi, K. and Nagai, S.-I. and Ninomiya-Tsuji, J. and Nishita, M. and Tamai, K. and Irie, K. and Ueno, N. and Nishida, E. and Shibuya, H. and Matsumoto, K.}, year={1999}, pages={179–187} }
 @article{nomoto_watanabe_ninomiya-tsuji_yang_kiuchi_hagiwara_hidaka_matsumoto_irie_1997, title={Functional analyses of mammalian protein kinase C isozymes in budding yeast and mammalian fibroblasts}, volume={2}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0031240976&partnerID=MN8TOARS}, number={10}, journal={Genes to Cells}, author={Nomoto, S. and Watanabe, Y. and Ninomiya-Tsuji, J. and Yang, L.-X. and Kiuchi, K. and Hagiwara, M. and Hidaka, H. and Matsumoto, K. and Irie, K.}, year={1997}, pages={601–614} }
 @article{ninomiya-tsuji_matsumoto_shibuya_1997, title={TGF-beta signaling}, volume={42}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0031183774&partnerID=MN8TOARS}, number={10 Suppl}, journal={Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme}, author={Ninomiya-Tsuji, J. and Matsumoto, K. and Shibuya, H.}, year={1997}, pages={1517–1524} }
 @article{ota_maeda_odashima_ninomiya-tsuji_tatsuka_1996, title={G<inf>1</inf> phase-specific suppression of the Cdk2 activity by ginsenoside Rh<inf>2</inf> in cultured murine cells}, volume={60}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0030636535&partnerID=MN8TOARS}, DOI={10.1016/s0024-3205(96)00608-x}, number={2}, journal={Life Sciences}, author={Ota, T. and Maeda, M. and Odashima, S. and Ninomiya-Tsuji, J. and Tatsuka, M.}, year={1996} }
 @article{tsuji_ninomiya-tsuji_torti_torti_1993, title={Augmentation by IL-1α of tumor necrosis factor-α cytotoxicity in cells transfected with adenovirus E1A}, volume={150}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0027223371&partnerID=MN8TOARS}, number={5}, journal={Journal of Immunology}, author={Tsuji, Y. and Ninomiya-Tsuji, J. and Torti, S.V. and Torti, F.M.}, year={1993}, pages={1897–1907} }
 @article{tsuji_ninomiya-tsuji_torti_torti_1993, title={Selective loss of CDC2 and CDK2 induction by tumor necrosis factor-α in senescent human diploid fibroblasts}, volume={209}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0027146392&partnerID=MN8TOARS}, number={2}, journal={Experimental Cell Research}, author={Tsuji, Y. and Ninomiya-Tsuji, J. and Torti, S.V. and Torti, F.M.}, year={1993}, pages={175–182} }
 @article{ninomiya-tsuji_torti_ringold_1993, title={Tumor necrosis factor-induced c-myc expression in the absence of mitogenesis is associated with inhibition of adipocyte differentiation}, volume={90}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0027491022&partnerID=MN8TOARS}, number={20}, journal={Proceedings of the National Academy of Sciences of the United States of America}, author={Ninomiya-Tsuji, J. and Torti, F.M. and Ringold, G.M.}, year={1993}, pages={9611–9615} }
 @article{yasuda_nakata_kamijo_honda_nakamura_ninomiya-tsuji_yamashita_nagahama_ohba_1992, title={Cyclin-dependent kinase 2 (cdk2) in the murine cdc2 kinase TS mutant}, volume={18}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0027085917&partnerID=MN8TOARS}, DOI={10.1007/BF01233079}, number={5}, journal={Somatic Cell and Molecular Genetics}, author={Yasuda, H. and Nakata, T. and Kamijo, M. and Honda, R. and Nakamura, M. and Ninomiya-Tsuji, J. and Yamashita, M. and Nagahama, Y. and Ohba, Y.}, year={1992}, pages={403–408} }
 @article{shibuya_yoneyama_ninomiya-tsuji_matsumoto_taniguchi_1992, title={IL-2 and EGF receptors stimulate the hematopoietic cell cycle via different signaling pathways: Demonstration of a novel role for c-myc}, volume={70}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0026749324&partnerID=MN8TOARS}, DOI={10.1016/0092-8674(92)90533-I}, number={1}, journal={Cell}, author={Shibuya, H. and Yoneyama, M. and Ninomiya-Tsuji, J. and Matsumoto, K. and Taniguchi, T.}, year={1992}, pages={57–67} }
 @article{shibuya_irie_ninomiya-tsuji_goebl_taniguchi_matsumoto_1992, title={New human gene encoding a positive modulator of HIV Tat-mediated transactivation}, volume={357}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0026689020&partnerID=MN8TOARS}, number={6380}, journal={Nature}, author={Shibuya, H. and Irie, K. and Ninomiya-Tsuji, J. and Goebl, M. and Taniguchi, T. and Matsumoto, K.}, year={1992}, pages={700–702} }
 @article{ninomiya-tsuji_nomoto_yasuda_reed_matsumoto_1991, title={Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation}, volume={88}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0025946804&partnerID=MN8TOARS}, number={20}, journal={Proceedings of the National Academy of Sciences of the United States of America}, author={Ninomiya-Tsuji, J. and Nomoto, S. and Yasuda, H. and Reed, S.I. and Matsumoto, K.}, year={1991}, pages={9006–9010} }
 @article{takasuka_ninomiya-tsuji_sakayama_ishibashi_ide_1990, title={A temperature-sensitive cell-cycle mutant of mammalian cells, tsJT16, is defective in a function operating soon after growth stimulation}, volume={15}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0025276307&partnerID=MN8TOARS}, number={1}, journal={Cell Structure and Function}, author={Takasuka, T. and Ninomiya-Tsuji, J. and Sakayama, M. and Ishibashi, S. and Ide, T.}, year={1990}, pages={39–45} }
 @article{tai_ninomiya-tsuji_furuoku_ogawa_ishibashi_shiroki_segawa_tsuchida_shibuya_ide_1990, title={Nonlethal G<inf>0</inf>-ts mutant tsJT60 becomes lethal at the nonpermissive temperature after transformation: A hint for new cancer chemotherapeutics}, volume={15}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0025572529&partnerID=MN8TOARS}, DOI={10.1247/csf.15.385}, abstractNote={tsJT60 is a nonlethal temperature-sensitive (ts) mutant of a Fischer rat cell line (3Y1) classified as a G0 mutant; i.e., the ts defect is not expressed within the cell growth cycle but is expressed only between the G0 and S phase. tsJT60 clones transformed with oncogenes such as adenovirus E1A, polyoma large T, polyoma middle T, v-Ki-ras, and LTR activated c-myc, or with a chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, grew well at 34°C. However, most of these clones grew slowly at 40°C, producing many floating dead cells, and some clones were killed at 40°C. When they were cultured under conditions inadequate for growth of untransformed cells, such as high cell density or serum restriction, they were killed at 40°C. These and previous results from SV40- and adenovirus-transformed tsJT60 clones favour the idea that transformed tsJT60 cells occasionally enter the G0 phase and are metabolically imbalanced at 40°C during self-stimulation from the G0 to S phase. We propose that a drug which exclusively block, G0-G1 transition would be cytocidal to transformed cells but cytostatic to normal cells.}, number={6}, journal={Cell Structure and Function}, author={Tai, Y.Y. and Ninomiya-Tsuji, J. and Furuoku, K. and Ogawa, N. and Ishibashi, S. and Shiroki, K. and Segawa, K. and Tsuchida, N. and Shibuya, M. and Ide, T.}, year={1990}, pages={385–391} }
 @article{tai_goto_ninomiya-tsuji_kameoka_ishibashi_shiroki_ide_1988, title={A cell cycle G<inf>0</inf>-ts mutant, tsJT60, becomes lethal at the nonpermissive temperature after transformation with adenovirus 12 E1B 19K mutant}, volume={179}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0023692566&partnerID=MN8TOARS}, DOI={10.1016/0014-4827(88)90347-3}, number={1}, journal={Experimental Cell Research}, author={Tai, Y.Y. and Goto, Y. and Ninomiya-Tsuji, J. and Kameoka, Y. and Ishibashi, S. and Shiroki, K. and Ide, T.}, year={1988}, pages={50–57} }
 @article{ninomiya-tsuji_nakahara_ito_akiyama_ishibashi_ide_1987, title={Bypass of the ts Block of tsJT60, a G<inf>0</inf>-Specific ts Mutant from Rat Fibroblasts, by Fetal Bovine Serum and Epidermal Growth Factor}, volume={12}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0023623034&partnerID=MN8TOARS}, DOI={10.1247/csf.12.421}, abstractNote={tsJT60, a temperature-sensitive (ts) G0-mutant cell line from a Fischer rat, grows normally in the exponential growth phase at 34 degrees C and 39.5 degrees C, but when stimulated with fetal bovine serum (FBS), from the G0 phase they reenter the S phase at 34 degrees C but not at 39.5 degrees C. The ts-block was bypassed when G0-arrested tsJT60 cells were stimulated at 39.5 degrees C with FBS plus epidermal growth factor (EGF). The presence of EGF for the first 6 h after serum stimulation caused tsJT60 cells to enter the S phase in the presence of FBS at 39.5 degrees C. When EGF was added 6 h after serum stimulation, entrance into the S phase was delayed by about 6 h. The sequential presence of two growth factors, EGF without FBS for 6 h then FBS without EGF, or the reversed sequence, failed to initiate DNA synthesis at 39.5 degrees C. The binding of EGF was not temperature sensitive. The amounts of RNA and protein present doubled after stimulation with both FBS and EGF at 39.5 degrees C. These and other findings suggest that EGF bypasses only some specific event in the entire prereplicative process that operates operating in serum-stimulated cells at 39.5 degrees C.}, number={5}, journal={Cell Structure and Function}, author={Ninomiya-Tsuji, J. and Nakahara, Y. and Ito, C. and Akiyama, T. and Ishibashi, S. and Ide, T.}, year={1987}, pages={421–432} }
 @article{miura_ninomiya-tsuji_tsuji_ishibashi_ide_1987, title={Colchicine activates cell cycle-dependent genes in growth-arrested rat 3Y1 cells}, volume={173}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0023514467&partnerID=MN8TOARS}, DOI={10.1016/0014-4827(87)90356-9}, number={1}, journal={Experimental Cell Research}, author={Miura, M. and Ninomiya-Tsuji, J. and Tsuji, Y. and Ishibashi, S. and Ide, T.}, year={1987}, pages={294–298} }
 @article{ninomiya-tsuji_ishibashi_ide_1987, title={Entrance of SV40-transformed Cells into g0 Phase as Revealed by a Study Using the g0-specific ts mutant tsjt60}, volume={47}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0023589313&partnerID=MN8TOARS}, number={22}, journal={Cancer Research}, author={Ninomiya-Tsuji, J. and Ishibashi, S. and Ide, T.}, year={1987}, pages={6028–6032} }
 @article{ninomiya-tsuji_nakahara_ito_akiyama_ishibashi_ide_1987, title={Epidermal growth factor has a unique effect in combination with fetal bovine serum to bypass the ts-block of G<inf>o</inf>-specific ts mutant tsJT60}, volume={171}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0023242003&partnerID=MN8TOARS}, DOI={10.1016/0014-4827(87)90253-9}, number={1}, journal={Experimental Cell Research}, author={Ninomiya-Tsuji, J. and Nakahara, Y. and Ito, C. and Akiyama, T. and Ishibashi, S. and Ide, T.}, year={1987}, pages={86–93} }
 @article{ninomiya-tsuji_goto_ishibashi_shiroki_ide_1987, title={Induction of cellular DNA synthesis in G<inf>0</inf>-specific ts mutant, tsJT60, following Infection with SV40 and adenoviruses}, volume={171}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0023501857&partnerID=MN8TOARS}, DOI={10.1016/0014-4827(87)90183-2}, number={2}, journal={Experimental Cell Research}, author={Ninomiya-Tsuji, J. and Goto, Y. and Ishibashi, S. and Shiroki, K. and Ide, T.}, year={1987}, pages={509–512} }
 @article{goto_ninomiya-tsuji_tanonaka_ishibashi_shiroki_ide_1987, title={tsJT60, a cell cycle G0-ts mutant, becomes lethal at non-permissive temperature by transformation with adenovirus 5 when the expression of E1B gene is lacking}, volume={170}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0023257723&partnerID=MN8TOARS}, DOI={10.1016/0014-4827(87)90323-5}, number={2}, journal={Experimental Cell Research}, author={Goto, Y. and Ninomiya-Tsuji, J. and Tanonaka, K. and Ishibashi, S. and Shiroki, K. and Ide, T.}, year={1987}, pages={491–498} }
 @article{ninomiya-tsuji_goto_ishibashi_ide_1986, title={Defect in prereplicative phase of G0-specific ts mutant, tsJT60}, volume={165}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0022547766&partnerID=MN8TOARS}, DOI={10.1016/0014-4827(86)90543-4}, number={1}, journal={Experimental Cell Research}, author={Ninomiya-Tsuji, J. and Goto, Y. and Ishibashi, S. and Ide, T.}, year={1986}, pages={191–198} }
 @article{ide_ninomiya-tsuji_ferrari_philiponis_baserga_1986, title={Expression of Growth-Regulated Genes in tsJT60 Cells, a Temperature-Sensitive Mutant of the Cell Cycle}, volume={25}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0022993107&partnerID=MN8TOARS}, DOI={10.1021/bi00370a043}, abstractNote={We have investigated the expression of growth-regulated genes in tsJT60 cells, a temperature-sensitive (ts) mutant of Fischer rat cells, which, on the basis of its kinetic behavior, can be classified as a G0 mutant. It grows normally at 34 degrees C and also at 39.5 degrees C if shifted to the higher temperature during exponential growth. However, if the cell population is first made quiescent by serum deprivation, subsequent stimulation by serum induces the cells to enter S phase at 34 degrees C but not at 39.5 degrees C. A panel of growth-regulated genes was used that included three protooncogenes (c-fos, c-myc, and p53), several genes that are induced in G0 cells stimulated by growth factors (beta-actin, 2A9, 2F1, vimentin, JE-3, KC-1, and ornithine decarboxylase), and an S-phase gene (histone H3). The expression of these growth-regulated genes was studied in both tsJT60 cells and its parental cell line, rat 3Y1 cells. All the genes tested, except histone H3, are similarly induced when quiescent tsJT60 cells are stimulated by serum at either permissive or restrictive temperatures. These results raise intriguing questions on the nature of quiescence and the relationship between G0 and G1 in cells in culture.}, number={22}, journal={Biochemistry}, author={Ide, T. and Ninomiya-Tsuji, J. and Ferrari, S. and Philiponis, V. and Baserga, R.}, year={1986}, pages={7041–7046} }
 @article{kihara_ninomiya-tsuji_ishibashi_ide_1986, title={Failure in S6 protein phosphorylation by serum stimulatio of senescent human diploid fibroblasts, TIG-1}, volume={37}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0022968467&partnerID=MN8TOARS}, DOI={10.1016/0047-6374(86)90115-6}, number={1}, journal={Mechanisms of Ageing and Development}, author={Kihara, F. and Ninomiya-Tsuji, J. and Ishibashi, S. and Ide, T.}, year={1986}, pages={27–40} }
 @article{tanonaka_ninomiya-tsuji_ishibashi_ide_1986, title={Isolation of ts mutant cells which arrest in G1/G0 phase at the non-permissive temperature in the presence of appropriate growth factors from a Fischer rat cell line, 3Y1}, volume={165}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0022508457&partnerID=MN8TOARS}, DOI={10.1016/0014-4827(86)90587-2}, number={2}, journal={Experimental Cell Research}, author={Tanonaka, K. and Ninomiya-Tsuji, J. and Ishibashi, S. and Ide, T.}, year={1986}, pages={337–344} }
 @article{ide_ninomiya_ishibashi_1984, title={Isolation of a G0-specific ts mutant from a Fischer rat cell line, 3Y1}, volume={150}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-0021328860&partnerID=MN8TOARS}, DOI={10.1016/0014-4827(84)90701-8}, number={1}, journal={Experimental Cell Research}, author={Ide, T. and Ninomiya, J. and Ishibashi, S.}, year={1984}, pages={60–67} }
 @article{isolation of a g0-specific ts mutant from a fischer rat cell line, 3y1. _1984, journal={Exp. Cell Res.}, year={1984} }