@article{mitra_chen_dhandaydham_wang_blackburn_kota_goshe_schwartz_huber_clouse_2015, title={An autophosphorylation site database for leucine-rich repeat receptor-like kinases in Arabidopsis thaliana}, volume={82}, ISSN={["1365-313X"]}, DOI={10.1111/tpj.12863}, abstractNote={SummaryLeucine‐rich repeat receptor‐like kinases (LRR RLKs) form a large family of plant signaling proteins consisting of an extracellular domain connected by a single‐pass transmembrane sequence to a cytoplasmic kinase domain. Autophosphorylation on specific Ser and/or Thr residues in the cytoplasmic domain is often critical for the activation of several LRR RLK family members with proven functional roles in plant growth regulation, morphogenesis, disease resistance, and stress responses. While identification and functional characterization of in vivo phosphorylation sites is ultimately required for a full understanding of LRR RLK biology and function, bacterial expression of recombinant LRR RLK cytoplasmic catalytic domains for identification of in vitro autophosphorylation sites provides a useful resource for further targeted identification and functional analysis of in vivo sites. In this study we employed high‐throughput cloning and a variety of mass spectrometry approaches to generate an autophosphorylation site database representative of more than 30% of the approximately 223 LRR RLKs in Arabidopsis thaliana. We used His‐tagged constructs of complete cytoplasmic domains to identify a total of 592 phosphorylation events across 73 LRR RLKs, with 497 sites uniquely assigned to specific Ser (268 sites) or Thr (229 sites) residues in 68 LRR RLKs. Multiple autophosphorylation sites per LRR RLK were the norm, with an average of seven sites per cytoplasmic domain, while some proteins showed more than 20 unique autophosphorylation sites. The database was used to analyze trends in the localization of phosphorylation sites across cytoplasmic kinase subdomains and to derive a statistically significant sequence motif for phospho‐Ser autophosphorylation.}, number={6}, journal={PLANT JOURNAL}, author={Mitra, Srijeet K. and Chen, Ruiqiang and Dhandaydham, Murali and Wang, Xiaofeng and Blackburn, Robert Kevin and Kota, Uma and Goshe, Michael B. and Schwartz, Daniel and Huber, Steven C. and Clouse, Steven D.}, year={2015}, month={Jun}, pages={1042–1060} } @article{halter_imkampe_mazzotta_wierzba_postel_buecherl_kiefer_stahl_chinchilla_wang_et al._2014, title={The Leucine-Rich Repeat Receptor Kinase BIR2 Is a Negative Regulator of BAK1 in Plant Immunity}, volume={24}, ISSN={["1879-0445"]}, DOI={10.1016/j.cub.2013.11.047}, abstractNote={BackgroundTransmembrane leucine-rich repeat (LRR) receptors are commonly used innate immune receptors in plants and animals but can also sense endogenous signals to regulate development. BAK1 is a plant LRR-receptor-like kinase (RLK) that interacts with several ligand-binding LRR-RLKs to positively regulate their functions. BAK1 is involved in brassinosteroid-dependent growth and development, innate immunity, and cell-death control by interacting with the brassinosteroid receptor BRI1, immune receptors, such as FLS2 and EFR, and the small receptor kinase BIR1, respectively.ResultsIdentification of in vivo BAK1 complex partners by LC/ESI-MS/MS uncovered two novel BAK1-interacting RLKs, BIR2 and BIR3. Phosphorylation studies revealed that BIR2 is unidirectionally phosphorylated by BAK1 and that the interaction between BAK1 and BIR2 is kinase-activity dependent. Functional analyses of bir2 mutants show differential impact on BAK1-regulated processes, such as hyperresponsiveness to pathogen-associated molecular patterns (PAMP), enhanced cell death, and resistance to bacterial pathogens, but have no effect on brassinosteroid-regulated growth. BIR2 interacts constitutively with BAK1, thereby preventing interaction with the ligand-binding LRR-RLK FLS2. PAMP perception leads to BIR2 release from the BAK1 complex and enables the recruitment of BAK1 into the FLS2 complex.ConclusionsOur results provide evidence for a new regulatory mechanism for innate immune receptors with BIR2 acting as a negative regulator of PAMP-triggered immunity by limiting BAK1-receptor complex formation in the absence of ligands.}, number={2}, journal={CURRENT BIOLOGY}, author={Halter, Thierry and Imkampe, Julia and Mazzotta, Sara and Wierzba, Michael and Postel, Sandra and Buecherl, Christoph and Kiefer, Christian and Stahl, Mark and Chinchilla, Delphine and Wang, Xiaofeng and et al.}, year={2014}, month={Jan}, pages={134–143} } @article{bajwa_wang_blackburn_goshe_mitra_williams_bishop_krasnyanski_allen_huber_et al._2013, title={Identification and Functional Analysis of Tomato BRI1 and BAK1 Receptor Kinase Phosphorylation Sites}, volume={163}, ISSN={["1532-2548"]}, DOI={10.1104/pp.113.221465}, abstractNote={Abstract Brassinosteroids (BRs) are plant hormones that are perceived at the cell surface by a membrane-bound receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1). BRI1 interacts with BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) to initiate a signal transduction pathway in which autophosphorylation and transphosphorylation of BRI1 and BAK1, as well as phosphorylation of multiple downstream substrates, play critical roles. Detailed mechanisms of BR signaling have been examined in Arabidopsis (Arabidopsis thaliana), but the role of BRI1 and BAK1 phosphorylation in crop plants is unknown. As a foundation for understanding the mechanism of BR signaling in tomato (Solanum lycopersicum), we used liquid chromatography-tandem mass spectrometry to identify multiple in vitro phosphorylation sites of the tomato BRI1 and BAK1 cytoplasmic domains. Kinase assays showed that both tomato BRI1 and BAK1 are active in autophosphorylation as well as transphosphorylation of each other and specific peptide substrates with a defined sequence motif. Site-directed mutagenesis revealed that the highly conserved kinase domain activation loop residue threonine-1054 was essential for tomato BRI1 autophosphorylation and peptide substrate phosphorylation in vitro. Furthermore, analysis of transgenic lines expressing full-length tomato BRI1-Flag constructs in the weak tomato bri1 allele, curl3-abs1, demonstrated that threonine-1054 is also essential for normal BRI1 signaling and tomato growth in planta. Finally, we cloned the tomato ortholog of TGF-β Receptor Interacting Protein (TRIP1), which was previously shown to be a BRI1-interacting protein and kinase domain substrate in Arabidopsis, and found that tomato TRIP1 is a substrate of both tomato BRI1 and BAK1 kinases in vitro.}, number={1}, journal={PLANT PHYSIOLOGY}, author={Bajwa, Vikramjit S. and Wang, Xiaofeng and Blackburn, R. Kevin and Goshe, Michael B. and Mitra, Srijeet K. and Williams, Elisabeth L. and Bishop, Gerard J. and Krasnyanski, Sergei and Allen, George and Huber, Steven C. and et al.}, year={2013}, month={Sep}, pages={30–42} } @article{oh_wang_clouse_huber_2012, title={Deactivation of the Arabidopsis BRASSINOSTEROID INSENSITIVE 1 (BRI1) receptor kinase by autophosphorylation within the glycine-rich loop}, volume={109}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.1108321109}, abstractNote={ The activity of the dual-specificity receptor kinase, brassinosteroid insensitive 1 (BRI1), reflects the balance between phosphorylation-dependent activation and several potential mechanisms for deactivation of the receptor. In the present report, we elucidate a unique mechanism for deactivation that involves autophosphorylation of serine-891 in the ATP-binding domain. Serine-891 was identified previously as a potential site of autophosphorylation by mass spectrometry, and sequence-specific antibodies and mutagenesis studies now unambiguously establish phosphorylation of this residue. In vivo, phosphorylation of serine-891 increased slowly with time following application of brassinolide (BL) to Arabidopsis seedlings, whereas phosphorylation of threonine residues increased rapidly and then remained constant. Transgenic plants expressing the BRI1(S891A)–Flag-directed mutant have increased hypocotyl and petiole lengths, relative to wild-type BRI1–Flag (both in the bri1-5 background), and accumulate higher levels of the unphosphorylated form of the BES1 transcription factor in response to exogenous BL. In contrast, plants expressing the phosphomimetic S891D-directed mutant are severely dwarfed and do not accumulate unphosphorylated BES1 in response to BL. Collectively, these results suggest that autophosphorylation of serine-891 is one of the deactivation mechanisms that inhibit BRI1 activity and BR signaling in vivo. Many arginine-aspartate (RD)-type leucine-rich repeat receptor-like kinases have a phosphorylatable residue within the ATP-binding domain, suggesting that this mechanism may play a broad role in receptor kinase deactivation. }, number={1}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Oh, Man-Ho and Wang, Xiaofeng and Clouse, Steven D. and Huber, Steven C.}, year={2012}, month={Jan}, pages={327–332} } @article{oh_wang_wu_zhao_clouse_huber_2010, title={Autophosphorylation of Tyr-610 in the receptor kinase BAK1 plays a role in brassinosteroid signaling and basal defense gene expression (Retracted article. See vol. 113, pg. E3987, 2016)}, volume={107}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0915064107}, abstractNote={ BAK1 is a leucine-rich repeat receptor-like kinase that functions as a coreceptor with the brassinosteroid (BR) receptor BRI1 and the flagellin receptor FLS2, and as a negative regulator of programmed cell death. BAK1 has been shown to autophosphorylate on numerous serine/threonine sites in vitro as well as to transphosphorylate associated receptor kinases both in vitro and in planta. In the present study we identify Tyr-610 in the carboxyl-terminal domain of BAK1 as a major site of autophosphorylation that is brassinolide-induced in vivo and requires a kinase-active BAK1. Expression of BAK1(Y610F)-Flag in transgenic plants lacking the endogenous bak1 and its functional paralogue, bkk1 , produced plants that were viable but extremely small and generally resembled BR signaling mutants, whereas an acidic substitution for Tyr-610 to mimic phosphorylation restored normal growth. Several lines of evidence support the notion that BR signaling is impaired in the BAK1(Y610F)-Flag plants, and are consistent with the recently proposed sequential transphosphorylation model for BRI1/BAK1 interaction and activation. In contrast, the FLS2-mediated inhibition of seedling growth by the flg22 elicitor occurred normally in the Y610F-directed mutant. However, expression of many defense genes was dramatically reduced in BAK1(Y610F) plants and the nonpathogenic hrpA mutant of Pseudomonas syringae was able to grow rapidly in the mutant. These results indicate that phosphorylation of Tyr-610 is required for some but not all functions of BAK1, and adds significantly to the emerging notion that tyrosine phosphorylation could play an important role in plant receptor kinase signaling. }, number={41}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Oh, Man-Ho and Wang, Xiaofeng and Wu, Xia and Zhao, Youfu and Clouse, Steven D. and Huber, Steven C.}, year={2010}, month={Oct}, pages={17827–17832} } @article{oh_wang_kota_goshe_clouse_huber_2009, title={Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis}, volume={106}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0810249106}, abstractNote={ Brassinosteroids (BRs) are essential growth-promoting hormones that regulate many aspects of plant growth and development. Two leucine-rich repeat receptor-like kinases (LRR-RLKs) are involved in BR perception and signal transduction: brassinosteroid insensitive 1 (BRI1), which is the BR receptor, and its coreceptor BRI1-associated kinase 1 (BAK1). Both proteins are classified as serine/threonine protein kinases, but here we report that recombinant cytoplasmic domains of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases. With BRI1, Tyr-831 and Tyr-956 are identified as autophosphorylation sites in vitro and in vivo. Interestingly, Tyr-956 in kinase subdomain V is essential for activity, because the Y956F mutant is catalytically inactive and thus this site cannot be simply manipulated by mutagenesis. In contrast, Tyr-831 in the juxtamembrane domain is not essential for kinase activity but plays an important role in BR signaling in vivo, because expression of BRI1(Y831F)-Flag in transgenic bri1–5 plants results in plants with larger leaves (but altered leaf shape) and early flowering relative to plants expressing wild-type BRI1-Flag. Acidic substitutions of Tyr-831 restored normal leaf size (but not shape) and normal flowering time. This is an example where a specific tyrosine residue has been shown to play an important role in vivo in plant receptor kinase function. Interestingly, 6 additional LRR-RLKs (of the 23 tested) were also found to autophosphorylate on tyrosine in addition to serine and threonine, suggesting that tyrosine signaling should be considered with other plant receptor kinases as well. }, number={2}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Oh, Man-Ho and Wang, Xiaofeng and Kota, Uma and Goshe, Michael B. and Clouse, Steven D. and Huber, Steven C.}, year={2009}, month={Jan}, pages={658–663} } @article{li_li_wang_yu_2009, title={Denial of service attacks and defenses in decentralized trust management}, volume={8}, ISSN={["1615-5270"]}, DOI={10.1007/s10207-008-0068-8}, abstractNote={Trust management is an approach to scalable and flexible access control in decentralized systems. In trust management, a server often needs to evaluate a chain of credentials submitted by a client; this requires the server to perform multiple expensive digital signature verifications. In this paper, we study low-bandwidth Denial-of-Service (DoS) attacks that exploit the existence of trust management systems to deplete server resources. Although the threat of DoS attacks has been studied for some application-level protocols such as authentication protocols, we show that it is especially destructive for trust management systems. Exploiting the delegation feature in trust management languages, an attacker can forge a long credential chain to force a server to consume a large amount of computing resource. Using game theory as an analytic tool, we demonstrate that unprotected trust management servers will easily fall prey to a witty attacker who moves smartly. We report our empirical study of existing trust management systems, which manifests the gravity of this threat. We also propose a defense technique using credential caching, and show that it is effective in the presence of intelligent attackers.}, number={2}, journal={INTERNATIONAL JOURNAL OF INFORMATION SECURITY}, author={Li, Jiangtao and Li, Ninghui and Wang, XiaoFeng and Yu, Ting}, year={2009}, month={Apr}, pages={89–101} } @article{wang_kota_he_blackburn_li_goshe_huber_clouse_2008, title={Sequential transphosphorylation of the BRI1/BAK1 receptor kinase complex impacts early events in brassinosteroid signaling}, volume={15}, ISSN={["1534-5807"]}, DOI={10.1016/j.devcel.2008.06.011}, abstractNote={Brassinosteroids (BRs) regulate plant development through a signal transduction pathway involving the BRI1 and BAK1 transmembrane receptor kinases. The detailed molecular mechanisms of phosphorylation, kinase activation, and oligomerization of the BRI1/BAK1 complex in response to BRs are uncertain. We demonstrate that BR-dependent activation of BRI1 precedes association with BAK1 in planta, and that BRI1 positively regulates BAK1 phosphorylation levels in vivo. BRI1 transphosphorylates BAK1 in vitro on specific kinase-domain residues critical for BAK1 function. BAK1 also transphosphorylates BRI1, thereby quantitatively increasing BRI1 kinase activity toward a specific substrate. We propose a sequential transphosphorylation model in which BRI1 controls signaling specificity by direct BR binding followed by substrate phosphorylation. The coreceptor BAK1 is then activated by BRI1-dependent transphosphorylation and subsequently enhances signaling output through reciprocal BRI1 transphosphorylation. This model suggests both conservation and distinct differences between the molecular mechanisms regulating phosphorylation-dependent kinase activation in plant and animal receptor kinases.}, number={2}, journal={DEVELOPMENTAL CELL}, author={Wang, Xiaofeng and Kota, Uma and He, Kai and Blackburn, Kevin and Li, Jia and Goshe, Michael B. and Huber, Steven C. and Clouse, Steven D.}, year={2008}, month={Aug}, pages={220–235} } @article{wu_wang_2006, title={Effects of plant growth regulator 2,4-D, KT and BA on callus induction and plant regeneration from mature embryos of maize}, number={80}, journal={Maize Genetics Cooperation Newsletter}, author={Wu, M. S. and Wang, X. F.}, year={2006}, pages={25} } @article{wang_goshe_soderblom_phinney_kuchar_li_asami_yoshida_huber_clouse_2005, title={Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase}, volume={17}, ISSN={["1532-298X"]}, DOI={10.1105/tpc.105.031393}, abstractNote={Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography–tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.}, number={6}, journal={PLANT CELL}, author={Wang, XF and Goshe, MB and Soderblom, EJ and Phinney, BS and Kuchar, JA and Li, J and Asami, T and Yoshida, S and Huber, SC and Clouse, SD}, year={2005}, month={Jun}, pages={1685–1703} }