@article{benson_christensen_fairchild_davis_2009, title={The mRNA for zona pellucida proteins B1, C and D in two genetic lines of turkey hens that differ in fertility}, volume={111}, ISSN={["1873-2232"]}, DOI={10.1016/j.anireprosci.2008.02.013}, abstractNote={The avian inner perivitelline layer (IPVL) contains zona pellucida protein-B1 (ZPB1), zona pellucida protein-C (ZPC) and zona pellucida protein-D (ZPD). These three proteins may be involved in sperm binding to the IPVL. ZPB1 is produced by the liver and transported to the developing preovulatory follicle, while ZPC and ZPD are synthesized and secreted by the granulosa cells of the preovulatory follicle. The mRNA of ZPB1, ZPC, and ZPD was investigated in two lines of turkey hens selected for over 40 generations for either increased egg production (E line) or increased body weight (F line). Total RNA was extracted from the liver and from 1cm(2) sections of the granulosa layer around the germinal disc and a nongerminal disc area of the F(1) and F(2) follicles of hens from each genetic line. Northern analysis was performed using chicken cDNA probes for all three ZP proteins. Hepatic mRNA for ZPB1 was greater (P<0.05) in turkey hens from the E line than the F line. Although, there was no difference in ZPC mRNA between the germinal disc and nongerminal disc region of the two largest follicles in E line hens, ZPC mRNA was greater in the nongerminal disc region compared to the germinal disc region in the two largest follicles obtained from the F line hens. There were no differences in ZPD mRNA between the germinal disc and nongerminal disc regions of the F(1) and F(2) follicles for either genetic line. The results suggest that the greater rates of fertility previously observed in eggs from the E line hens compared with the F line of hens may be related to differential amounts of the potential sperm binding proteins ZPB1 and ZPC.}, number={2-4}, journal={ANIMAL REPRODUCTION SCIENCE}, author={Benson, A. P. and Christensen, V. L. and Fairchild, B. D. and Davis, A. J.}, year={2009}, month={Apr}, pages={149–159} }
 @article{davis_im_dubin_tomer_boss_2007, title={Arabidopsis phosphatidylinositol phosphate kinase 1 binds F-actin and recruits phosphatidylinositol 4-kinase beta 1 to the actin cytoskeleton (Retracted article. See vol. 284, pg. 16060, 2009)}, volume={282}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M611728200}, abstractNote={The actin cytoskeleton can be influenced by phospholipids and lipid-modifying enzymes. In animals the phosphatidylinositol phosphate kinases (PIPKs) are associated with the cytoskeleton through a scaffold of proteins; however, in plants such an interaction was not clear. Our approach was to determine which of the plant PIPKs interact with actin and determine whether the PIPK-actin interaction is direct. Our results indicate that AtPIPK1 interacts directly with actin and that the binding is mediated through a predicted linker region in the lipid kinase. AtPIPK1 also recruits AtPI4Kβ1 to the cytoskeleton. Recruitment of AtPI4Kβ1 to F-actin was dependent on the C-terminal catalytic domain of phosphatidylinositol-4-phosphate 5-kinase but did not require the presence of the N-terminal 251 amino acids, which includes 7 putative membrane occupation and recognition nexus motifs. In vivo studies confirm the interaction of plant lipid kinases with the cytoskeleton and suggest a role for actin in targeting PIPKs to the membrane. The actin cytoskeleton can be influenced by phospholipids and lipid-modifying enzymes. In animals the phosphatidylinositol phosphate kinases (PIPKs) are associated with the cytoskeleton through a scaffold of proteins; however, in plants such an interaction was not clear. Our approach was to determine which of the plant PIPKs interact with actin and determine whether the PIPK-actin interaction is direct. Our results indicate that AtPIPK1 interacts directly with actin and that the binding is mediated through a predicted linker region in the lipid kinase. AtPIPK1 also recruits AtPI4Kβ1 to the cytoskeleton. Recruitment of AtPI4Kβ1 to F-actin was dependent on the C-terminal catalytic domain of phosphatidylinositol-4-phosphate 5-kinase but did not require the presence of the N-terminal 251 amino acids, which includes 7 putative membrane occupation and recognition nexus motifs. In vivo studies confirm the interaction of plant lipid kinases with the cytoskeleton and suggest a role for actin in targeting PIPKs to the membrane. Arabidopsis phosphatidylinositol phosphate kinase 1 binds F-actin and recruits phosphatidylinositol 4-kinase β1 to the actin cytoskeleton.Journal of Biological ChemistryVol. 284Issue 23PreviewVOLUME 282 (2007) PAGES 14121–14131 Full-Text PDF Open Access Phosphatidylinositol phosphate kinases (PIPKs) 2The abbreviations used are: PIPK, phosphatidylinositol phosphate kinase; eEF1A, eukaryotic elongation factor 1A; MORN, membrane occupation and recognition nexus; PtdIns4P, phosphatidylinositol-4-phosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; PI4K, phosphatidylinositol kinase; PtdOH, phosphatidic acid; MALDI, matrix-assisted laser desorption ionization time; PLC, phospholipase C; GST, glutathione S-transferase; MS, mass spectroscopy; PIPES, 1,4-piperazinediethanesulfonic acid. are a family of enzymes that phosphorylate phosphatidylinositol phosphates (PtdInsP) to phosphatidylinositol bisphosphates (PtdInsP2). Arabidopsis has 11 predicted isoforms of PtdInsP kinases (1Mueller-Roeber B. Pical C. Plant Physiol. 2002; 130: 22-46Crossref PubMed Scopus (322) Google Scholar). AtPIPK1 and AtPIPK10 have been characterized biochemically, and PtdIns4P is the primary substrate making the predominant product phosphatidylinositol (4Perera I.Y. Davis A.J. Galanopoulou D. Im Y.J. Boss W.F. FEBS Lett. 2005; 579: 3427-3432Crossref PubMed Scopus (52) Google Scholar, 5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) bisphosphate (PtdIns(4,5)P2), suggesting that they are similar to the type I PIPKs found in humans (2Hinchliffe K.A. Ciruela A. Irvine R.F. Biochim. Biophys. Acta. 1998; 1436: 87-104Crossref PubMed Scopus (102) Google Scholar, 3Westergren T. Dove S.K. Sommarin M. Pical C. Biochem. J. 2001; 359: 583-589Crossref PubMed Scopus (48) Google Scholar, 4Perera I.Y. Davis A.J. Galanopoulou D. Im Y.J. Boss W.F. FEBS Lett. 2005; 579: 3427-3432Crossref PubMed Scopus (52) Google Scholar, 5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). However, unlike the mammalian, yeast or Caenorhabditis elegans PIPKs, AtPIPK1–9 contain N-terminal putative membrane occupation and recognition nexus (MORN) motifs similar to those first reported in junctophilins (1Mueller-Roeber B. Pical C. Plant Physiol. 2002; 130: 22-46Crossref PubMed Scopus (322) Google Scholar, 6Takeshima H. Komazaki S. Nishi M. Lino M. Kangawa K. Mol. Cell. 2000; 6: 11-22Abstract Full Text Full Text PDF PubMed Google Scholar). MORN motifs are unique to this family of enzymes and have not been reported in any other eukaryotic lipid kinases. In eukaryotic models PtdInsP kinases supply PtdIns(4,5)P2 for many cellular functions, and it is well known that phospholipids play an integral role in regulating the structure and dynamics of the cytoskeleton through the many actin-binding proteins that interact with PtdIns(4,5)P2 (7Simonsen A. Wurmser A.E. Emr S.D. Stenmark H. Curr. Opin. Cell Biol. 2001; 13: 485-492Crossref PubMed Scopus (407) Google Scholar, 8Takenawa T. Itoh T. Biochim. Biophys. Acta. 2001; 1533: 190-206Crossref PubMed Scopus (245) Google Scholar, 9Downes C.P. Gray A. Lucocq J.M. Trends Cell Biol. 2005; 15: 259-268Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 10Yin H.L. Janmey P.A. Annu. Rev. Physiol. 2002; 65: 761-789Crossref PubMed Scopus (567) Google Scholar). For example, the absence of PtdInsP kinases in yeast has an adverse affect on the yeast cell morphology (11Homma K. Terui S. Minemura M. Qadota H. Anraku Y. Kanaho Y. Ohya Y. J. Biol. Chem. 1998; 273: 15779-15786Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and causes them to be unable to properly form actin cables (12Desrivieres S. Cooke F.T. Parker P.J. Hall M.N. J. Biol. Chem. 1998; 271: 15787-15793Abstract Full Text Full Text PDF Scopus (177) Google Scholar). In some animal cell lines overexpression of PtdInsP kinases results in the formation of actin comet tails and stress fibers (13Yamamoto M. Hilgemann D.H. Feng S. Bito H. Ishihara H. Shibasaki Y. Yin H.L. J. Cell Biol. 2001; 152: 867-876Crossref PubMed Scopus (104) Google Scholar, 14Kanzaki M. Furukawa M. Raab W. Pessin J.E. J. Biol. Chem. 2004; 279: 30622-30633Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and disruption of membrane trafficking (14Kanzaki M. Furukawa M. Raab W. Pessin J.E. J. Biol. Chem. 2004; 279: 30622-30633Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In these animal cell lines, a functional PtdInsP kinase producing PtdIns(4,5)P2 was necessary to cause the changes in cytoskeletal structure (13Yamamoto M. Hilgemann D.H. Feng S. Bito H. Ishihara H. Shibasaki Y. Yin H.L. J. Cell Biol. 2001; 152: 867-876Crossref PubMed Scopus (104) Google Scholar, 15Tolias K.F. Hartwig J.H. Ishihara H. Shibasaki Y. Cantlry L.C. Carpenter C.L. Curr. Biol. 2000; 10: 153-156Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). In mammalian systems, PIPK activity has been recovered with an F-actin fraction, and the α, β, and γ isoforms have been identified as co-purifying with F-actin. The data support a model where these PIPKs do not directly interact with F-actin but that this interaction is mediated by the presence of Racs, small GTP-binding proteins (16Yang S.A. Carpenter C.L. Abrams C.S. J. Biol. Chem. 2004; 279: 42331-42336Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In plants, PtdIns kinase activity (17Xu P. Lloyd C.W. Staiger C.J. Drøbak B.K. Plant Cell. 1992; 4: 941-951Crossref PubMed Google Scholar, 18Tan Z. Boss W.F. Plant Physiol. 1992; 100: 2116-2120Crossref PubMed Scopus (73) Google Scholar) and PtdInsP kinase activity co-purify with an F-actin-enriched fraction (18Tan Z. Boss W.F. Plant Physiol. 1992; 100: 2116-2120Crossref PubMed Scopus (73) Google Scholar), but it is not clear from these studies whether plant PtdInsP kinases directly bind F-actin or, like animal PIPKs, associate with a scaffold of actin-binding proteins. Actin remodeling in root hairs and pollen tubes, sites of rapid growth in plants, is sensitive to alterations in PtdIns(4,5)P2 biosynthesis. It was first noted that expression of mutant Arabidopsis Rac2 in tobacco pollen tubes decreased plasma membrane PtdIns(4,5)P2 and disrupted the normal actin filament orientation (19Kost B. Lemichez E. Spielhofer P. Hong Y. Tolias K. Carpenter C. Chua N.H. J. Cell Biol. 1999; 145: 317-330Crossref PubMed Scopus (444) Google Scholar). The GFP-AtRac2 was localized to the apical tip of the pollen tube, and PtdInsP kinase activity was co-immunoprecipitated with Rac2 antibodies. More recent work in pollen tubes suggested that phospholipase C-mediated PtdIns(4,5)P2 turnover also affected actin structure and suggested that PtdIns(4,5)P2 metabolism is necessary in normal pollen growth (20Dowd P.E. Coursol S. Skirpan A.L. Kao T.H. Gilroy S. Plant Cell. 2006; 18: 1438-1453Crossref PubMed Scopus (183) Google Scholar, 21Helling D. Possart A. Cottier S. Klahre U. Kost B. Plant Cell. 2006; 18: 3519-3534Crossref PubMed Scopus (193) Google Scholar). Expression of inactive mutants of PLC isoforms resulted in increased levels PtdIns(4,5)P2 throughout the entire pollen tube and enriched at the apical tip (20Dowd P.E. Coursol S. Skirpan A.L. Kao T.H. Gilroy S. Plant Cell. 2006; 18: 1438-1453Crossref PubMed Scopus (183) Google Scholar, 21Helling D. Possart A. Cottier S. Klahre U. Kost B. Plant Cell. 2006; 18: 3519-3534Crossref PubMed Scopus (193) Google Scholar). In both tobacco (21Helling D. Possart A. Cottier S. Klahre U. Kost B. Plant Cell. 2006; 18: 3519-3534Crossref PubMed Scopus (193) Google Scholar) and petunia (20Dowd P.E. Coursol S. Skirpan A.L. Kao T.H. Gilroy S. Plant Cell. 2006; 18: 1438-1453Crossref PubMed Scopus (183) Google Scholar), disruption of PLC hydrolysis of PtdIns(4,5)P2 using inactive mutants or chemical inhibitors of PLC resulted in tip swelling, suggesting that PLC-mediated PtdIns(4,5)P2 turnover was essential for normal tip-directed growth. In root hairs, decreased tip-localized PtdIns(4,5)P2 resulting from a loss of the inositol phospholipid transfer protein, Atsfh1p, also resulted in unfocused root hair growth and disruption of the tip-directed actin filament orientation (22Vincent P. Chua M. Nogue F. Fairbrother A. Mekeel H. Xu Y. Allen N. Bibikova T.N. Gilroy S. Bankaitis V.A. J. Cell Biol. 2005; 168: 801-812Crossref PubMed Scopus (168) Google Scholar). Both in pollen and root hairs the data suggested that tip-localized PtdIns(4,5)P2 affected tip growth via F-actin-mediated vesicle trafficking (23Boss W.F. Davis A.J. Im Y.J. Galvao R.M. Perera I.Y. Subcell. Biochem. 2006; 39: 181-205Crossref PubMed Scopus (28) Google Scholar). Such a role for the inositol lipids in actin-mediated vesicle trafficking was supported by the work of Preuss et al. (24Preuss M.L. Serna J. Falbel T.G. Bednarek S.Y. Nielsen E. Plant Cell. 2004; 16: 1589-1603Crossref PubMed Scopus (200) Google Scholar). They showed that mutants in PI4Kβ1 and PI4Kβ2 or treatment with latrunculin (24Preuss M.L. Serna J. Falbel T.G. Bednarek S.Y. Nielsen E. Plant Cell. 2004; 16: 1589-1603Crossref PubMed Scopus (200) Google Scholar) disrupted trafficking of specialized RabA4b-associated Golgi vesicles in root hairs. Blocking turnover of PtdIns(4,5)P2 and increasing PtdIns(4,5)P2 pools by a mutation in inositol lipid phosphatases also affected actin filament orientation and cell wall biosynthesis in inflorescent stems of Arabidopsis (25Zhong R. Burk D.H. Morrison W.H. II I Ye Z.H. Plant Cell. 2004; 16: 3242-3259Crossref PubMed Scopus (99) Google Scholar). Although all of these data strongly support a role for PtdIns(4,5)P2 regulating actin cytoskeleton and vesicle trafficking in plants, the underlying mechanism is not known. Because the major family of plant PIPKs have a very different structure to the PIPKs of animals and yeast, it is important to understand how they are regulated and to identify their interacting partners. In this work our approach was to identify proteins that interact specifically with AtPIPK1 and AtPIPK10 and to determine whether one of the PIPKs would associate with F-actin. We show that Arabidopsis PIPK1 directly interacts with actin and recruits PI4Kβ1, suggesting a plant-specific mechanism for influencing cytoskeletal dynamics and lipid signaling. Because AtPIPK1 has an N-terminal MORN domain with the potential for tight membrane adhesion, the presence of AtPIPK1 on F-actin would provide a discrete pool of PtdIns(4,5,)P2 for actin-mediated vesicle trafficking and/or fusion. Production of GST Fusion Proteins in Escherichia coli—GST-AtPIPK1 and GST-AtPIPK10 were cloned and expressed in E. coli as previously described (4Perera I.Y. Davis A.J. Galanopoulou D. Im Y.J. Boss W.F. FEBS Lett. 2005; 579: 3427-3432Crossref PubMed Scopus (52) Google Scholar, 5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). AtPI4Kβ1 was PCR amplified using Pfx DNA polymerase with forward primer 5′-CACCATGCCGATGGGACGCTTT-3′ and reverse primer 5′-CTCACACTCTTCCATTTAAGACCCGTTGGTA-3′. The resulting PCR product was subcloned into the pENTR/SD/D-TOPO destination vectors (Invitrogen) and then into pDEST15 for expression of GST-AtPI4Kβ1 proteins in E. coli. The inactive form of AtPIPK1, AtPIPK1(K468A), was mutated in the ATP binding site using QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA). The ATP binding site was identified by sequence homology to that of the Human PtdInsP kinase, HsPIPK1α, and mutated as described (26Park S.J. Itoh T. Takenawa T. J. Biol. Chem. 2001; 276: 4781-4787Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The mutated construct was amplified with forward primer 5′-CTCAAGATGATAGATTTATGATCGCAACGGTGAAGAAATCAGAAGTCAAG-3′ and reverse primer 5′-CTTGACTTCTGATTTCTTCACCGTTGCGATCATAAATCTATCATCTTGAG-3′. The resulting PCR product was subcloned into the pENTR/SD/D-TOPO destination vectors (Invitrogen) and then into pDEST15 for expression of GST-AtPIPK1(K468A). Truncations of AtPIPK1, MORN, ΔMORN, ΔMORN/ΔL, ΔMORN/ΔC, and ΔMORN/ΔL/ΔC were produced as previously described (27Im Y.J. Davis A.J. Perera I.Y. Johannes E. Allen N.S. Boss W.F. J. Biol. Chem. 2007; 282: 5443-5452Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). All GST fusion proteins were produced and purified as previously described (5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Protein concentration was determined using the Bio-Rad protein assay reagent with bovine serum albumin as a standard. Purified recombinant protein bound to the glutathione-Sepharose beads was stored at 4 °C until use in protein binding or activity assays. Plant Material—The recombinant binary plasmid pK7WGF2-HsPIPK1α was transformed into Agrobacterium tumefaciens EHA105 by the freeze-thaw method. For stable transformation, NT-1 cells were transformed using A. tumefaciens-mediated gene transfer following the protocol of Perera et al. (4Perera I.Y. Davis A.J. Galanopoulou D. Im Y.J. Boss W.F. FEBS Lett. 2005; 579: 3427-3432Crossref PubMed Scopus (52) Google Scholar). Cells were subcultured weekly into 25 ml of NT-1 culture medium containing 50 μgml–1 kanamycin as described by Perera et al. (4Perera I.Y. Davis A.J. Galanopoulou D. Im Y.J. Boss W.F. FEBS Lett. 2005; 579: 3427-3432Crossref PubMed Scopus (52) Google Scholar). NT-1 cells expressing ΔMORN were produced as previously described (27Im Y.J. Davis A.J. Perera I.Y. Johannes E. Allen N.S. Boss W.F. J. Biol. Chem. 2007; 282: 5443-5452Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Protein Pulldown Assays—Protein pulldown assays were performed with purified recombinant protein incubated with precleared Triton-solubilized Arabidopsis membrane fractions for 2 h at 4 °C with continuous mixing in 30 mm β-cyclodextrin, phosphate-buffered saline (0.1 m KH2PO4, 0.1 m K2HPO4, 135 mm NaCl, and 2.7 mm KCl, pH 7.3), and final concentrations of 3 mm ATP or 0.5 mm GTP where indicated. Triton-solubilized Arabidopsis membranes were prepared by incubating a 40,000 × g pellet isolated as described previously in 1% (v/v) Triton X-100 10 min at 4 °C then centrifuging at 10,000 × g for 10 min to obtain a Triton-solubilized supernatant (5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Membrane fractions were precleared by incubating the solubilized membranes with purified GST immobilized on glutathione-Sepharose beads. The cleared supernatant was used for protein-protein interactions. After incubation with the membrane proteins, the beads were washed extensively with phosphate-buffered saline. Direct interactions of proteins were investigated using purified recombinant proteins either coupled to beads, released from the beads with reduced glutathione (Novagen, manufacturers instructions), or cleaved from the GST tag with thrombin (Novagen, manufacturers instructions) in phosphate-buffered saline or where indicated by using purified native protein. After washing unbound proteins from the beads, the bound proteins and beads were either added directly into 4× SDS-PAGE sample buffer, heated at 100 °C for 5 min for separation by SDS-PAGE, or were used immediately for lipid kinase activity analysis. Mass Spectrometry—After SDS-PAGE, the gels were stained with Coomassie Brilliant Blue, and stained bands were selected for excision. Digestion was carried out with bands excised from 10% polyacrylamide gel stained with Coomassie in 20 ml of 100 mm ammonium bicarbonate buffer, pH 8.0, 1 mm CaCl2. 1 ml of modified trypsin (sequencing grade, Promega) solution (2 mg/ml in 50 mm acetic acid) was added to yield a final enzyme protein ratio of 1:10. After incubation at 37 °C for 2 h, 2 ml of 1 n HCl was added to bring the mixture to pH 3.0 and inactivate the trypsin. One-half of the sample (10 ml) was moved to another tube and mixed with 2 ml of 0.1 m Tris-(2-carboxyethyl)phosphin (Pierce) in 0.1 m citrate buffer, pH 3.0. The mixture was incubated at 37 °C for 30 min for reduction of the disulfide bond-containing tryptic peptides. 1-ml aliquots of reduced or non-reduced tryptic peptide mixtures were used for MALDI-MS analysis without further purification. MALDI time-of-flight MS analyses were carried out using a Voyager-DE STR mass spectrometer (Applied Biosystems Inc.) equipped with a pulsed UV nitrogen laser (337 nm, 3-ns pulse) and a dual microchannel plate detector. For molecular mass determination of peptides, spectra were acquired at linear-delayed extraction (DE) mode, acceleration voltage set at 25 kV, grid voltage at 95% of the acceleration voltage, delay time at 320 ns, and low mass gate set at 1000 Da. The mass to charge ratio was calibrated with the molecular mass of a mixture of proteins (mass, 5,734.58–16,952.56). For analysis of tryptic peptides, the spectra were acquired at reflectron-DE mode with acceleration voltage set to 20 kV, grid voltage at 72% of the acceleration voltage, delay time at 200 ns, and low mass gate at 250 Da. The mass to charge ratio was calibrated with the mass of a mixture of standard peptides (mass, 904.46–5,734.58). Saturated α-cyano-4-hydroxycinnamic acid in 70% acetonitrile containing 0.1% trifluoroacetic acid was used as the matrix for analysis of tryptic peptides, and saturated sinapinic acid in 50% acetonitrile containing 0.1% trifluoroacetic acid was used as the matrix for protein analysis. 1 μl of a solution of reduced or non-reduced tryptic peptide mixture was applied on the MALDI plate followed by 1 μl of saturated matrix solution. Spectra were recorded after evaporation of the solvent and processed using Data Explorer software for data collection and analysis. Predicted masses were calculated by the ExPASy Peptide Mass program. Immunoblotting Blotting—After separation by SDS-PAGE, proteins were transferred to polyvinylidene difluoride membranes by electroblotting. Membranes were blocked in 5% (w/v) powdered milk in Tris-buffered saline. Blots were incubated with the primary antibody for 1 h followed by incubation in the secondary antibody for 1 h. Secondary antibodies were coupled to horseradish peroxidase or to IRDye800 (Pierce). For antibodies coupled to horseradish peroxidase, immunoreactivity was visualized by incubating the blot in SuperSignal West Pico Chemiluminescent substrate (Pierce) and exposure to x-ray film. For the fluorescently labeled secondary antibodies, immunoreactivity was detected with Odyssey infrared imaging system (Licor, Lincoln NE) according to the manufacturer’s instructions. After immunoreactivity detection, total protein was visualized by staining the blots with Amido Black (Sigma). Lipid Kinase Assays—PtdInsP kinase activity and phosphatidylinositol kinase activity was assayed in duplicate as described by (5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) with a final reaction volume of 50 μl. Each assay contained 10 μg of purified recombinant protein on glutathione-Sepharose beads that had been incubated with solubilized Arabidopsis membrane fraction or with purified proteins and washed once with 50 mm Tris, pH 7.5. Lipid substrate was prepared using PtdIns4P or PtdIns (porcine brain; Avanti Polar Lipids, Alabaster, AL) from 1 or 5 mg/ml stocks, respectively. Lipids were dried under an N2 atmosphere and solubilized for use in the lipid kinase assays in the presence of cyclodextrins as described previously (5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The lipid kinase assay was performed as described (5Davis A.J. Perera I.Y. Boss W.F. J. Lipid Res. 2004; 45: 1783-1789Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Lipid extraction was performed using an acid solvent system. Extracted lipids were separated by TLC on silica gel plates (LK5D; Whatman, Clifton, NJ) using a CHCl3:MeOH: NH4OH:water (90:90:7:22, v/v) solvent system. The 32P-labeled phospholipids were quantified with a Bioscan System 500 imaging scanner. Actin Polymerization—Polymerization buffer contained 20 mm PIPES, 2 mm EGTA, 2 mm MgCl2, 1 mm ATP, 50 mm KCl, pH 6.5 (28Carraway K.L. Carraway C.A.C. The Cytoskeleton: A Practical Approach. 1992; (Oxford University Press, New York): 47-71Google Scholar). Buffer was added to 30 μg of protein from the depolymerized actin fraction from Arabidopsis membranes, 5 μg of plasma membrane isolated from NT-1 cells as described previously (29Heilmann I. Perera I.Y. Gross W. Boss W.F. Plant Physiol. 1999; 229: 1331-1339Crossref Scopus (43) Google Scholar), or to 3 μg of pure actin containing purified recombinant proteins eluted from glutathione-Sepharose beads with reduced glutathione. Polymerization reactions were incubated for 1 h at 25 °C, then the F-actin was pelleted by centrifugation. For centrifugation at 20,000 × g for 1 h at 4 °C, polymerization volume was 30 μl. For centrifugation at 100,000 × g for 30 min, polymerization volume was 100 μl (28Carraway K.L. Carraway C.A.C. The Cytoskeleton: A Practical Approach. 1992; (Oxford University Press, New York): 47-71Google Scholar). Latrunculin treatments were performed by adding 10 μm latrunculin B to 2 gof 4-day-old NT-1 cells for 1 h (30Van Gestel K. Kohler R.H. Verbelen J.P. J. Exp. Bot. 2002; 53: 659-667Crossref PubMed Scopus (186) Google Scholar). An equal volume of Me2SO was used as a control. Cells were harvested, and the plasma membrane was isolated as previously described (29Heilmann I. Perera I.Y. Gross W. Boss W.F. Plant Physiol. 1999; 229: 1331-1339Crossref Scopus (43) Google Scholar). Insect Cell Protein Production—Serum-free Spodoptera frugiperda (Sf9) cells were obtained from Invitrogen and maintained at 28 °C at a concentration of 2.5 × 105 to 5 × 106 cells ml–1 in Sf-900 II insect cell serum-free medium (Invitrogen). The expression system used was the Bac-to-Bac baculovirus expression system (Invitrogen). Recombinant baculoviruses were generated from the recombinant expression vectors according to the manufacturer’s recommendations. Optimal production of AtPI4Kβ1 was 2 days after infection, and optimal production of AtPI4Kα1 (31Stevenson-Paulik J. Love J. Boss W.F. Plant Physiol. 2003; 132: 1053-1064Crossref PubMed Scopus (32) Google Scholar) and AtPI4Kγ7(ΔN/ΔC) was on the third day after infection. 3R. M. Galvąo and W. F. Boss, unpublished results. All assays were performed using cells optimally producing the respective polypeptide. Infected cells were then harvested and lysed as previously described (31Stevenson-Paulik J. Love J. Boss W.F. Plant Physiol. 2003; 132: 1053-1064Crossref PubMed Scopus (32) Google Scholar). The cleared lysate was analyzed for protein concentration by the Bradford method (Bio-Rad) and used for protein-protein interactions. Cyclodextrin Enhanced Recovery of Proteins in Pulldown Reactions—Our first goal was to identify proteins that interacted specifically with AtPIPK1. For this purpose, constructs were developed to produce GST fusion proteins of AtPIPK1 and AtPIPK10 in E. coli to pulldown-interacting proteins from Arabidopsis cell fractions. Fig. 1 shows the production of the recombinant GST-tagged proteins in E. coli as detected by a GST antibody (Fig. 1A) and with antibodies raised against AtPIPK1 or AtPIPK10 (Fig. 1B). Although the antibodies raised against full-length AtPIPK1 and AtPIPK10 readily detected the recombinant E. coli-expressed proteins, these antibodies were unable to detect PtdInsP kinases from an Arabidopsis cell fraction (data not shown), suggesting that the antibodies were not robust and/or that the protein levels were very low. For this reason, we used GST antibodies or activity assays to monitor the enzymes. Because PtdInsP 5-kinase activity was associated with F-actin as well as plasma membranes from plants (18Tan Z. Boss W.F. Plant Physiol. 1992; 100: 2116-2120Crossref PubMed Scopus (73) Google Scholar, 29Heilmann I. Perera I.Y. Gross W. Boss W.F. Plant Physiol. 1999; 229: 1331-1339Crossref Scopus (43) Google Scholar, 32Sommarin M. Sandelius A.S. Biochim. Biophys. Acta. 1988; 958: 268-278Crossref Scopus (81) Google Scholar), to increase the recovery of potential interacting proteins, cells were homogenized using a buffer developed to enhance the recovery of F-actin with the membranes (33Abe S. Ito Y. Davies E. J. Exp. Bot. 1992; 43: 941-949Crossref Scopus (36) Google Scholar). Recovery of interacting proteins was compared using a 40,000 × g pellet and the soluble fraction from 4-day-old Arabidopsis cells grown in suspension culture. As described under “Experimental Procedures,” all fractions isolated from the suspension culture cells were pre-cleared by incubation with purified GST immobilized on glutathione-Sepharose beads to reduce the number of GST binding proteins. No interacting proteins were evident based on Coomassie and silver staining when the 40,000 × g supernatant was used for the pulldown assays with either GST-AtPIPK1 or GST-AtPIPK10 (data not shown); however, several proteins were evident if the Triton X-100-solubilized membrane fraction was used. Although there were interacting proteins present for both isoforms from the solubilized membrane fraction, there was not enough protein present on the stained gels to identify the proteins by mass spectrometry. To enhance the recovery of the solubilized proteins, we developed a protocol that involved adding cyclodextrins to the pulldown reaction mixture. Cyclodextrins have been used to aid in protein refolding and in sequestering lipids (34Fauvelle F. Debouzy J.C. Crouzy S. Goschl M. Chapron Y. J. Pharm. Sci. 1997; 86: 935-943Abstract Full Text PDF PubMed Scopus (66) Google Scholar, 35Karuppiah N. Sharma A. Biochem. Biophys. Res. Commun. 1995; 211: 60-66Crossref PubMed Scopus (134) Google Scholar). α-, β-, and γ-cyclodextrins (0–50 mm) were added after pre-clearing the Triton-solubilized membrane proteins to see which if any would enhance the recovery of interacting proteins. γ-Cyclodextrin did not improve the interaction of the Arabidopsis proteins with the recombinant proteins at any of the concentrations tested, and α-cyclodextrin enhanced the interactions of a few of the proteins from the membranes with the recombinant proteins (data not shown); however, when 30 mm β-cyclodextrin was added, there was a 6-fold increase in the recovery of the peptides detected (Fig. 1C). One possible explanation for the enhanced protein recovery is that the cyclodextrin helps the recombinant protein fold properly to allow for interaction with the Arabidopsis peptides (35Karuppiah N. Sharma A. Biochem. Biophys. Res. Commun. 1995; 211: 60-66Crossref PubMed Scopus (134) Google Scholar). In addition, cyclodextrin will sequester the Triton or lipids present in the membrane fraction and thereby may enhance the interaction of the protein partners during the pulldown reaction. Because β-cyclodextrin enhanced recovery and did not appear to affect the distribution or number of the proteins bands, it was used for all subsequent pulldown experiments using microsomal proteins unless indicated otherwise. AtPIPK1 and AtPIPK10 Recovered Different Proteins Based on Mass Spectrometry Analysis—Using the optimized pull-down conditions for experiments, we were able to recover sufficient amounts of interacting peptides to identify them by mass spectrometry. To eliminate any false positives, we selected only for Coomassie-}, number={19}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Davis, Amanda J. and Im, Yang Ju and Dubin, Joshua S. and Tomer, Kenneth B. and Boss, Wendy F.}, year={2007}, month={May}, pages={14121–14131} }
 @article{im_perera_brglez_davis_stevenson-paulik_phillippy_johannes_allen_boss_2007, title={Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum}, volume={19}, ISSN={["1532-298X"]}, DOI={10.1105/tpc.107.051367}, abstractNote={Abstract}, number={5}, journal={PLANT CELL}, author={Im, Yang Ju and Perera, Imara Y. and Brglez, Irena and Davis, Amanda J. and Stevenson-Paulik, Jill and Phillippy, Brian Q. and Johannes, Eva and Allen, Nina S. and Boss, Wendy F.}, year={2007}, month={May}, pages={1603–1616} }
 @article{im_davis_perera_johannes_allen_boss_2007, title={The N-terminal membrane occupation and recognition nexus domain of Arabidopsis phosphatidylinositol phosphate kinase 1 regulates enzyme activity}, volume={282}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M611342200}, abstractNote={The type I B family of phosphatidylinositol phosphate kinases (PIPKs) contain a characteristic region of Membrane Occupation and Recognition Nexus (MORN) motifs at the N terminus. These MORN motifs are not found in PIPKs from other eukaryotes. To understand the impact of the additional N-terminal domain on protein function and subcellular distribution, we expressed truncated and full-length versions of AtPIPK1, one member of this family of PIPKs, in Escherichia coli and in tobacco cells grown in suspension culture. Deletion of the N-terminal MORN domain (amino acids 1–251) of AtPIPK1 increased the specific activity of the remaining C-terminal peptide (ΔMORN) >4-fold and eliminated activation by phosphatidic acid (PtdOH). PtdOH activation could also be eliminated by mutating Pro396 to Ala (P396A) in the predicted linker region between the MORN and the kinase homology domains. AtPIPK1 is product-activated and the MORN domain binds PtdIns(4,5)P2. Adding back the MORN peptide to ΔMORN or to the PtdOH-activated full-length protein increased activity ∼2-fold. Furthermore, expressing the MORN domain in vivo increased the plasma membrane PtdInsP kinase activity. When cells were exposed to hyperosmotic stress, the MORN peptide redistributed from the plasma membrane to a lower phase or endomembrane fraction. In addition, endogenous PtdInsP kinase activity increased in the endomembrane fraction of hyperosmotically stressed cells. We conclude that the MORN peptide can regulate both the function and distribution of the enzyme in a manner that is sensitive to the lipid environment.}, number={8}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Im, Yang Ju and Davis, Amanda J. and Perera, Imara Y. and Johannes, Eva and Allen, Nina S. and Boss, Wendy F.}, year={2007}, month={Feb}, pages={5443–5452} }
 @article{perera_davis_galanopilou_im_boss_2005, title={Characterization and comparative analysis of Arabidopsis phosphatidylinositol phosphate 5-kinase 10 reveals differences in Arabidopsis and human phosphatidylinositol phosphate kinases}, volume={579}, ISSN={["1873-3468"]}, DOI={10.1016/j.febslet.2005.05.018}, abstractNote={
Arabidopsis phosphatidylinositol phosphate (PtdInsP) kinase 10 (AtPIPK10; At4g01190) is shown to be a functional enzyme of the subfamily A, type I AtPtdInsP kinases. It is biochemically distinct from AtPIPK1 (At1g21980), the only other previously characterized AtPtdInsP kinase which is of the B subfamily. AtPIPK10 has the same K
m, but a 10‐fold lower V
max than AtPIPK1 and it is insensitive to phosphatidic acid. AtPIPK10 transcript is most abundant in inflorescence stalks and flowers, whereas AtPIPK1 transcript is present in all tissues. Comparative analysis of recombinant AtPIPK10 and AtPIPK1 with recombinant HsPIPKIα reveals that the Arabidopsis enzymes have roughly 200‐ and 20‐fold lower V
max/K
m, respectively. These data reveal one explanation for the longstanding mystery of the relatively low phosphatidylinositol‐(4,5)‐bisphosphate:phosphatidylinositol‐4‐phosphate ratio in terrestrial plants.}, number={16}, journal={FEBS LETTERS}, author={Perera, IY and Davis, AJ and Galanopilou, D and Im, YJ and Boss, WF}, year={2005}, month={Jun}, pages={3427–3432} }
 @article{davis_perera_boss_2004, title={Cyclodextrins enhance recombinant phosphatidylinositol phosphate kinase activity}, volume={45}, ISSN={["1539-7262"]}, DOI={10.1194/jlr.D400005-JLR200}, abstractNote={Inositol lipid kinases have been studied extensively in both plant and animal systems. However, major limitations for in vitro studies of recombinant lipid kinases are the low specific activity and instability of the purified proteins. Our goal was to determine if cyclodextrins would provide an effective substrate delivery system and enhance the specific activity of lipid kinases. For these studies, we have used recombinant Arabidopsis thaliana phosphatidylinositol phosphate kinase 1 (At PIPK1). At PIPK1 was produced as a fusion protein with glutathione-S-transferase and purified on glutathione-Sepharose beads. A comparison of lipid kinase activity using substrate prepared in α-, β-, or γ-cyclodextrin indicated that β-cyclodextrin was most effective and enhanced lipid kinase activity 6-fold compared with substrate prepared in Triton X-100-mixed micelles. We have optimized reaction conditions and shown that product can be recovered from the cyclodextrin-treated recombinant protein, which reveals a potential method for automating the assay for pharmacological screening.}, number={9}, journal={JOURNAL OF LIPID RESEARCH}, author={Davis, AJ and Perera, IY and Boss, WF}, year={2004}, month={Sep}, pages={1783–1789} }