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• W2113024008 abstract "Phosphatidylinositol transfer protein α (PITPα) participates in the supply of phosphatidylinositol (PI) required for many cellular events including phospholipase C (PLC) β and γ signaling by G-protein-coupled receptors and receptor-tyrosine kinases, respectively. Protein kinase C has been known to modulate PLC signaling by G-protein-coupled receptors and receptor-tyrosine kinases, although the molecular target has not been identified in most instances. In each case phorbol myristate acetate pretreatment of HL60, HeLa, and COS-7 cells abrogated PLC stimulation by the agonists formyl-Met-Leu-Phe, ATP, and epidermal growth factor, respectively. Here we show that phosphorylation of PITPα at Ser166 resulted in inhibition of receptor-stimulated PLC activity. Ser166 is localized in a small pocket between the 165–172 loop and the rest of the protein and was not solvent-accessible in either the PI- or phosphatidylcholine-loaded structures of PITPα. To allow phosphorylation at Ser166, a distinct structural form is postulated, and mutation of Thr59 to alanine shifted the equilibrium to this form, which could be resolved on native PAGE. The elution profile observed by size exclusion chromatography of phosphorylated PITPα from rat brain or in vitro phosphorylated PITPα demonstrated that phosphorylated PITPα is structurally distinct from the non-phosphorylated form. Phosphorylated PITPα was unable to deliver its PI cargo, although it could deliver phosphatidylcholine. We conclude that the PITPα structure has to relax to allow access to the Ser166 site, and this may occur at the membrane surface where PI delivery is required for receptor-mediated PLC signaling. Phosphatidylinositol transfer protein α (PITPα) participates in the supply of phosphatidylinositol (PI) required for many cellular events including phospholipase C (PLC) β and γ signaling by G-protein-coupled receptors and receptor-tyrosine kinases, respectively. Protein kinase C has been known to modulate PLC signaling by G-protein-coupled receptors and receptor-tyrosine kinases, although the molecular target has not been identified in most instances. In each case phorbol myristate acetate pretreatment of HL60, HeLa, and COS-7 cells abrogated PLC stimulation by the agonists formyl-Met-Leu-Phe, ATP, and epidermal growth factor, respectively. Here we show that phosphorylation of PITPα at Ser166 resulted in inhibition of receptor-stimulated PLC activity. Ser166 is localized in a small pocket between the 165–172 loop and the rest of the protein and was not solvent-accessible in either the PI- or phosphatidylcholine-loaded structures of PITPα. To allow phosphorylation at Ser166, a distinct structural form is postulated, and mutation of Thr59 to alanine shifted the equilibrium to this form, which could be resolved on native PAGE. The elution profile observed by size exclusion chromatography of phosphorylated PITPα from rat brain or in vitro phosphorylated PITPα demonstrated that phosphorylated PITPα is structurally distinct from the non-phosphorylated form. Phosphorylated PITPα was unable to deliver its PI cargo, although it could deliver phosphatidylcholine. We conclude that the PITPα structure has to relax to allow access to the Ser166 site, and this may occur at the membrane surface where PI delivery is required for receptor-mediated PLC signaling. Phosphatidylinositol transfer proteins (PITPs) 1The abbreviations used are: PITP, phosphatidylinositol transfer protein; PI, phosphatidylinositol; PC, phosphatidylcholine; fMLP, formyl-Met-Leu-Phe; PMA, phorbol myristate acetate; PKC, protein kinase C; PLC, phospholipase C; HPLC, high performance liquid chromatography; GTPγS, guanosine 5′-(γ-thio)triphosphate; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; EGF, epidermal growth factor; PIPES, 1,4-piperazinediethanesulfonic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid; GFP, green fluorescent protein; IEF, isoelectric focusing; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight.1The abbreviations used are: PITP, phosphatidylinositol transfer protein; PI, phosphatidylinositol; PC, phosphatidylcholine; fMLP, formyl-Met-Leu-Phe; PMA, phorbol myristate acetate; PKC, protein kinase C; PLC, phospholipase C; HPLC, high performance liquid chromatography; GTPγS, guanosine 5′-(γ-thio)triphosphate; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; EGF, epidermal growth factor; PIPES, 1,4-piperazinediethanesulfonic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid; GFP, green fluorescent protein; IEF, isoelectric focusing; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight. are a family of lipid-binding proteins that transfer individual molecules of phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments (1Wirtz K.W.A. Biochem. J. 1997; 324: 353-360Crossref PubMed Scopus (174) Google Scholar). Originally identified as soluble proteins of ∼35 kDa, the family of PITP-related proteins has subsequently grown to include three subgroups of proteins all containing a PITP domain (2Hsuan J. Cockcroft S. Genome Biol. 2001; (http://genomebiology.com/1465-6906/2/REVIEWS3011)PubMed Google Scholar, 3Allen-Baume V. Segui B. Cockcroft S. FEBS Lett. 2002; 531: 74-80Crossref PubMed Scopus (70) Google Scholar): the classical PITPs α and β (35 kDa), the larger related proteins RdgBαI and -II (160 kDa), and the soluble RdgBβ protein (38 kDa). The yeast Sec14p and its related family members form a separate group of proteins that, although they share lipid binding properties and transfer function with the mammalian PITPs, have no sequence or structural similarity (4Sha B. Phillips S.E. Bankaitis V. Luo M. Nature. 1998; 391: 506-510Crossref PubMed Scopus (230) Google Scholar, 5Yoder M.D. Thomas L.M. Tremblay J.M. Oliver R.L. Yarbrough L.R. Helmkamp Jr., G.M. J. Biol. Chem. 2001; 276: 9246-9252Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 6Tilley S.J. Skippen A. Murray-Rust J. Swigart P. Stewart A. Morgan C.P. Cockcroft S. McDonald N.Q. Structure (Lond.). 2004; 12: 317-326Abstract Full Text PDF PubMed Scopus (79) Google Scholar, 7Schouten A. Agianian B. Westerman J. Kroon J. Wirtz K.W. Gros P. EMBO J. 2002; 21: 2117-2121Crossref PubMed Scopus (66) Google Scholar). PITPα and PITPβ are 77% identical (and 94% similar) in amino acid sequence and have been implicated in both signaling and membrane traffic (8Cunningham E. Thomas G.M. Ball A. Hiles I. Cockcroft S. Curr. Biol. 1995; 5: 775-783Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 9Fensome A. Cunningham E. Prosser S. Tan S.K. Swigart P. Thomas G. Hsuan J. Cockcroft S. Curr. Biol. 1996; 6: 730-738Abstract Full Text Full Text PDF PubMed Google Scholar, 10Kauffmann-Zeh A. Thomas G.M. Ball A. Prosser S. Cunningham E. Cockcroft S. Hsuan J.J. Science. 1995; 268: 1188-1190Crossref PubMed Scopus (160) Google Scholar, 11Ohashi M. Jan de Vries K. Frank R. Snoek G. Bankaitis V. Wirtz K. Huttner W.B. Nature. 1995; 377: 544-547Crossref PubMed Scopus (168) Google Scholar, 12Hay J.C. Martin T.F.J. Nature. 1993; 366: 572-575Crossref PubMed Scopus (306) Google Scholar, 13Kular G. Loubtchenkov M. Swigart P. Whatmore J. Ball A. Cockcroft S. Wetzker R. Biochem. J. 1997; 325: 299-301Crossref PubMed Scopus (49) Google Scholar, 14Jones S.M. Alb Jr., J.G. Phillips S.E. Bankaitis V.A. Howell K.E. J. Biol. Chem. 1998; 273: 10349-10354Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Biochemical studies involving reconstitution of cytosol-depleted cell preparations with crude cytosol have consistently identified PITP (α and β) as a reconstitution factor in phospholipase C (PLC)-mediated PI 4,5-bisphosphate hydrolysis, the synthesis of 3-phosphorylated lipids by phosphoinositide 3-kinases, regulated exocytosis, and the biogenesis of vesicles at the Golgi (8Cunningham E. Thomas G.M. Ball A. Hiles I. Cockcroft S. Curr. Biol. 1995; 5: 775-783Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 9Fensome A. Cunningham E. Prosser S. Tan S.K. Swigart P. Thomas G. Hsuan J. Cockcroft S. Curr. Biol. 1996; 6: 730-738Abstract Full Text Full Text PDF PubMed Google Scholar, 10Kauffmann-Zeh A. Thomas G.M. Ball A. Prosser S. Cunningham E. Cockcroft S. Hsuan J.J. Science. 1995; 268: 1188-1190Crossref PubMed Scopus (160) Google Scholar, 11Ohashi M. Jan de Vries K. Frank R. Snoek G. Bankaitis V. Wirtz K. Huttner W.B. Nature. 1995; 377: 544-547Crossref PubMed Scopus (168) Google Scholar, 12Hay J.C. Martin T.F.J. Nature. 1993; 366: 572-575Crossref PubMed Scopus (306) Google Scholar, 13Kular G. Loubtchenkov M. Swigart P. Whatmore J. Ball A. Cockcroft S. Wetzker R. Biochem. J. 1997; 325: 299-301Crossref PubMed Scopus (49) Google Scholar, 14Jones S.M. Alb Jr., J.G. Phillips S.E. Bankaitis V.A. Howell K.E. J. Biol. Chem. 1998; 273: 10349-10354Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Hay J.C. Fisette P.L. Jenkins G.H. Fukami K. Takenawa T. Anderson R.E. Martin T.F.J. Nature. 1995; 374: 173-177Crossref PubMed Scopus (448) Google Scholar, 16Panaretou C. Domin J. Cockcroft S. Waterfield M.D. J. Biol. Chem. 1997; 272: 2477-2485Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Although both PITP isoforms can be used interchangeably in the reconstitution assays, they are likely to have some distinct functions in vivo. Deletion of the PITPα or PITPβ genes give distinct phenotypes; deletion of the PITPα gene leads to neurodegeneration and early death, while deletion of PITPβ is embryonically lethal (17Hamilton B.A. Smith D.J. Mueller K.L. Kerrebrock A.W. Bronson R.T. van Berkel V. Daly M.J. Kroglyak L. Reeve M.P. Nernhauser J.L. Hawkins T.L. Rubin E.M. Lander E.S. Neuron. 1997; 18: 711-722Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 18Alb Jr., J.G. Phillips S.E. Rostand K. Cui X. Pinxteren J. Cotlin L. Manning T. Guo S. York J.D. Sontheimer J.F. Collawn J.F. Bankaitis V.A. Mol. Biol. Cell. 2002; 13: 739-754Crossref PubMed Scopus (60) Google Scholar). Studies using genetic manipulation have failed to clarify the role of PITPα in PLC signaling possibly because of the overlapping roles of these isoforms (19Cunningham E. Tan S.W. Swigart P. Hsuan J. Bankaitis V. Cockcroft S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6589-6593Crossref PubMed Scopus (99) Google Scholar, 20Larijani B. Allen-Baume V. Morgan C.P. Li M. Cockcroft S. Curr. Biol. 2003; 13: 78-84Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). The ability of PITPα and PITPβ to transfer PI makes these soluble proteins ideally suited for regulating the spatial and temporal provision of PI in specific membrane compartments or in the nucleus where it can be phosphorylated by specific lipid kinases (2Hsuan J. Cockcroft S. Genome Biol. 2001; (http://genomebiology.com/1465-6906/2/REVIEWS3011)PubMed Google Scholar, 21Cockcroft S. De Matteis M.A. J. Membr. Biol. 2001; 180: 187-194Crossref PubMed Scopus (69) Google Scholar). Recently we have reported the plasma membrane association of both PITPα and -β isoforms when cells are activated by EGF to stimulate phospholipase C activity (20Larijani B. Allen-Baume V. Morgan C.P. Li M. Cockcroft S. Curr. Biol. 2003; 13: 78-84Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Phosphorylated forms of PI play essential roles in many cellular processes. The major requirement is for PI 4,5-bisphosphate at the plasma membrane where it is a substrate for both PLC and phosphoinositide 3-kinases, enzymes whose activity is regulated by cell surface receptors. Additional roles include regulation of ion channels, enzymatic activity of phospholipase D, maintenance of the cytoskeleton, and recruitment of target proteins by interaction with their phosphoinositide-binding domains including PH (pleckstrin homology), PX (pho × homology), FYVE, and ENTH (epsin/N-terminal homology) (21Cockcroft S. De Matteis M.A. J. Membr. Biol. 2001; 180: 187-194Crossref PubMed Scopus (69) Google Scholar, 22Cullen P.J. Cozier G.E. Banting G. Mellor H. Curr. Biol. 2001; 11: R882-R893Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Proteins containing these domains appear to be involved in membrane traffic including endocytosis, exocytosis, and vesicle budding. Many previous studies have reported that activation of protein kinase C (PKC) attenuates receptor-coupled PLC activity thus providing a negative feedback signal to limit the magnitude and duration of receptor signaling. Despite numerous examples of such regulation, the target for PKC-mediated inhibition has not been identified (Ref. 23Yue C. Ku C.Y. Liu M. Simon M.I. Sanborn B.M. J. Biol. Chem. 2000; 275: 30220-30225Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar; for reviews, see Refs. 24Rana R.S. Hokin L.E. Physiol. Rev. 1990; 70: 115-164Crossref PubMed Scopus (431) Google Scholar and 25Rhee S.G. Choi K.D. J. Biol. Chem. 1992; 267: 12393-12396Abstract Full Text PDF PubMed Google Scholar). These studies include both the G-protein-regulated PLCβ family and receptor-tyrosine kinase-regulated PLCγ family. Additionally no single lipid kinase has yet been identified that provides a dedicated pool of PI 4,5-bisphosphate for PLC-mediated hydrolysis. There are altogether four PI 4-kinases (Type II p55 isoforms (α and β) and Type III PI 4-kinase α and PI 4-kinase β) and at least three phosphatidylinositol 4-phosphate 5-kinases (plus splice variants) (26Gehrmann T. Heilmeyer Jr., L.M. Eur. J. Biochem. 1998; 253: 357-370Crossref PubMed Scopus (109) Google Scholar, 27Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1315) Google Scholar). So far there has been no evidence for negative regulation by phosphorylation of any of the lipid kinases by PMA. The signal transducing system and the phospholipases are different depending on the receptor, and the only common molecule that participates in both G-protein-regulated systems and receptor-tyrosine kinases is PITPα (10Kauffmann-Zeh A. Thomas G.M. Ball A. Prosser S. Cunningham E. Cockcroft S. Hsuan J.J. Science. 1995; 268: 1188-1190Crossref PubMed Scopus (160) Google Scholar, 19Cunningham E. Tan S.W. Swigart P. Hsuan J. Bankaitis V. Cockcroft S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6589-6593Crossref PubMed Scopus (99) Google Scholar, 28Thomas G.M. Cunningham E. Fensome A. Ball A. Totty N.F. Troung O. Hsuan J.J. Cockcroft S. Cell. 1993; 74: 919-928Abstract Full Text PDF PubMed Scopus (188) Google Scholar). In this study we examined the possibility that phosphorylation of PITPα by PKC may be responsible for the negative feedback inhibition. While this study was in progress, van Tiel et al. (29van Tiel C.M. Westerman J. Paasman M. Wirtz K.W.A. Snoek G.T. J. Biol. Chem. 2000; 275: 21532-21538Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) reported that PITPα was phosphorylated on Ser166in vitro by PKC. In the present study, we identified Thr59 (minor) and Ser166 (major) as two residues that when phosphorylated are negative regulators of receptor-stimulated PLC signaling. Our results support a model in which a structurally distinct pool of PITPα is phosphorylated at the membrane, and this phosphorylated PITPα is unable to deliver its PI cargo in exchange for PC at the plasma membrane. Materials—All standard chemicals were obtained from Sigma. γ-Labeled [32P]ATP, 32Pi, [3H]inositol, and [14C]acetate were obtained from Amersham Biosciences. Rat brain PKC (catalog no. 539494) was purchased from Calbiochem. This preparation predominantly contains the conventional PKC isoforms α, β1, β2, and γ. Endoproteinase Glu-C (V8 protease) and trypsin (modified) were sequencing grade and were obtained from Roche Applied Science. The lipids used for exchange were dimyristoyl PC, egg yolk PC, and bovine brain PI all from Sigma. Phosphorylation of PITPα by PMA Treatment of HL60 Cells—HL60 cells (2 × 108 cells) were washed and resuspended in 2 ml of buffer (20 mm Tris, 137 mm NaCl, 3 mm KCl, 1 mm CaCl2, and 1 mm MgCl2, pH 7.4), and the cells were incubated for 1 h with 1 mCi of 32P-labeled sodium orthophosphate at 37 °C. The cells were washed, resuspended in 4 ml, and allowed to equilibrate for 15 min at 37 °C. 900 μl of the cells were then added to tubes containing 100 μl of PMA (100 nm final concentration), and the cells were incubated for 0, 30, 60, and 300 s at 37 °C. The reaction was stopped by addition of 500 μl of lysis buffer (1% Nonidet P-40, 50 mm PIPES, pH 6.8) supplemented with phosphatase inhibitors, and insoluble material was removed by centrifugation. PITPα was immunoprecipitated using 5F12 monoclonal antibody (30Prosser S. Sarra R. Swigart P. Ball A. Cockcroft S. Biochem. J. 1997; 324: 19-23Crossref PubMed Scopus (16) Google Scholar) coupled to Sepharose, and immunoprecipitates were washed five times in lysis buffer. The immunoprecipitates were resolved by SDS-PAGE, Western blotted, phosphorimaged, and probed using the monoclonal antibody 5F12. Analysis of Receptor-stimulated Phospholipase C in Intact Cells—For HL60 cells, confluent suspension cultures (106 cells/ml) were incubated with 300 μm dibutyryl cAMP to differentiate the cells for 48 h in Medium 199 in the presence of 1 μCi/ml [3H]inositol. HeLa cells and COS-7 cells were labeled overnight with 2 μCi/ml [3H]inositol in Medium 199 supplemented with 1.5% dialyzed fetal calf serum and 5 μg/ml insulin and transferrin. In all cases, the cells were washed with HEPES buffer (20 mm HEPES, 137 mm NaCl, 3 mm KCl, 5.6 mm glucose, 1 mg/ml bovine serum albumin, 10 mm LiCl, 1 mm CaCl2, and 1 mm MgCl2, pH 7.2) and incubated with agonist. For HL60 cells, the cells were stimulated with fMLP (1 μm) for the indicated times. COS-7 cells were stimulated with EGF (100 ng/ml), and HeLa cells were stimulated with ATP (1 mm) for 20 min. Cells were pretreated with PMA (100 nm) for 5 min prior to stimulation with the agonist where indicated. Inositol phosphates were measured as described previously (8Cunningham E. Thomas G.M. Ball A. Hiles I. Cockcroft S. Curr. Biol. 1995; 5: 775-783Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). Production of Polyclonal Antisera to Phosphorylated Ser166—Peptide synthesis and immunization were performed by Eurogentec (Seraing, Belgium). The peptide sequences CEDPAKFKSIKTGRGP and CEDPAKFKpSIKTGRGP (where pS is phosphoserine) were synthesized, and two rabbits were immunized with the latter phosphorylated peptide. (Cysteine was added at the N terminus to aid coupling for the immunization process.) The serum was first purified using a Protein G-agarose column (10 ml). Serum was filtered through a 0.45-μm filter, applied to the column at 1 ml/min, and then washed extensively with TBS (25 mm Tris, pH 7.4, 137 mm NaCl, 5 mm KCl), 0.02% NaN3. Bound antibody was eluted using 100 mm glycine, pH 2.5, and then neutralized with 1 m Tris, pH 7.4. (The Protein G column was then washed with 6 m urea before reuse). The eluted antibody was concentrated and desalted to TBS, 0.02% NaN3 (8 ml). Peptide (10 mg) was coupled to 1 ml of N-hydroxysuccinimideagarose columns (Amersham Biosciences) using the manufacturer's suggested coupling conditions. Two columns were made, a column containing the phosphorylated peptide and a second with the non-phosphorylated peptide. Protein G-purified immunoglobulins were purified using the peptide columns. Initially the Protein G-purified immunoglobulins were applied to the phosphopeptide column at 0.2 ml/min, and the flow-through was then reapplied (to recover losses). The column was then washed extensively with TBS, 0.02% NaN3, and the bound immunoglobulins were eluted with 100 mm glycine, pH 2.5 (3 ml) and immediately desalted to TBS/NaN3 (4 ml). The resultant immunoglobulins were tested on dot blots containing dilutions of the synthetic phospho- and non-phosphopeptides. The antibody was further purified by applying to the non-phosphopeptide column. Briefly 1 ml was applied to the column and allowed to stand at room temperature for 30 min. The non-bound immunoglobulin (containing phosphopeptide antibodies) was washed through with 1 ml of TBS and then reapplied to the column in 2 × 1-ml lots (leaving to stand for 30 min in between). The column was then washed with 1 ml of TBS resulting in 3 ml of non-bound immunoglobulin. This preparation, depleted of antibodies against the non-phosphorylated peptide, was then used for Western blots. The specificity of the phosphoantibody was validated using the following criteria. The antibody was tested using PITPα mutants where the phosphorylation site was mutated to alanine (Fig. 4B) and by competing antibody binding with prior incubation with phosphopeptide and λ-phosphatase (New England Biolabs) treatment of phosphorylated PITPα (data not known). Production of PITPα-specific Polyclonal Antisera—Antibodies were raised in two rabbits against a specific PITPα peptide C-terminal sequence (CMRQKDPVKGMTADD). Peptide synthesis and immunization was performed by Eurogentec. Cysteine was added at the N terminus to aid coupling for the immunization process. The antisera were checked against recombinant PITPβ to confirm that they were specific for PITPα. Phosphorylation of PITPα in Acutely Permeabilized Cells—HL60 cells were acutely permeabilized with 0.6 IU/ml streptolysin O in PIPES buffer (20 mm PIPES, 137 mm NaCl, 3 mm KCl, 1 mg/ml bovine serum albumin, 1 mg/ml glucose, pH 6.8) in the presence of 1 mm MgATP, 2 mm MgCl2, and calcium buffered with EGTA at pCa 7 or 5 (31Stutchfield J. Cockcroft S. Biochem. J. 1988; 250: 375-382Crossref PubMed Scopus (72) Google Scholar). His-tagged wild-type and T59A PITPα proteins were included as indicated. Phospholipase C was stimulated with 10 μm GTPγS (31Stutchfield J. Cockcroft S. Biochem. J. 1988; 250: 375-382Crossref PubMed Scopus (72) Google Scholar). PMA (100 nm) was added to the cells for 10 min prior to permeabilization. Following incubation at 37 °C for 20 min, cells were removed by centrifugation, and the proteins were recaptured using nitrilotriacetic acid-agarose (Qiagen) for 1 h at 4 °C. Bound proteins were extensively washed with PIPES buffer (containing no bovine serum albumin or glucose). Proteins were eluted with 500 mm imidazole, desalted to 20 mm Tris-HCl, pH 7.6, and concentrated. Quantities of each protein were estimated, and equal amounts were examined for phosphorylation by Western blotting. Preparation of Rat Brain Cytosol Enriched in PITPα—Rat brains (9.5 g) were homogenized in 19 ml of buffer (20 mm PIPES, pH 6.8, 137 mm NaCl, 3 mm KCl, 5 mm EGTA, 5 mm EDTA) supplemented with 1 ml of protease inhibitors (Sigma catalog no. P-2714). The homogenate was centrifuged to pellet membranes and insoluble material at 100,000 × g at 4 °C for 1 h. Cytosol (20 ml) was filtered through a 0.45-μm membrane and concentrated in an Amicon pressure filtration device with a 10-kDa membrane to 10 ml (16 mg of protein/ml). PITP was purified from 9 ml of this cytosol (total protein, 144 mg) by gel filtration using a Superdex-75 HR 26/60 column (Amersham Biosciences). 5-ml fractions were collected, and PITPα-containing fractions were located by Western blotting with PITPα- and with phospho-Ser166-specific antibodies. Fractions enriched in PITPα were pooled and concentrated for use in the in vitro phosphorylation assay. Phosphorylation of PITPα in Vitro—Partially purified rat brain PITPα (16 μg of protein) and 0.34 μg of thrombin-cleaved recombinant PITPα were phosphorylated with PKC (0.1 unit) in a volume of 50 μl containing 20 mm Tris·Cl, 5 mm MgCl2, 200 μm CaCl2, 100 μg/ml phosphatidylserine, 10 μg/ml diacylglycerol, 100 μm MgATP, 200 μCi–2 mCi/ml γ-labeled [32P]ATP (pH 7.5) for 1 h and analyzed by Western blotting. Recombinant PITPα protein was initially phosphorylated with the His tag remaining; however, it was discovered that the His tag was phosphorylated by PKC. The His tag was therefore removed by thrombin cleavage prior to PKC treatment. PITPα proteins were desalted into cleavage buffer (20 mm Tris, 150 mm NaCl, 2.5 mm CaCl2, pH 8.4) and incubated with 1 unit of thrombin/mg of PITP for 16 h at 25 °C. PITPα was repurified by size exclusion chromatography. Cleaved recombinant PITPα was phosphorylated with purified rat brain PKC (Calbiochem). PITPα was preincubated with PI vesicles (40-fold molar excess) for 5 min at 30 °C to exchange the bound ligand to PI from the bacterially derived phosphatidylglycerol (32Hara S. Swigart P. Jones D. Cockcroft S. J. Biol. Chem. 1997; 272: 14909-14913Google Scholar). The mixture was phosphorylated with 70–350 ng (0.1–0.5 units) of PKC/5–10 μg of PITP in a volume of 50 μl containing 20 mm Tris·Cl, 5 mm MgCl2, 200 μm CaCl2, 100 μg/ml phosphatidylserine, 10 μg/ml diacylglycerol, 100 μm MgATP, 200 μCi–2 mCi/ml γ-labeled [32P]ATP (pH 7.5) at 30 °C for 20 min or 1 h as indicated. Phosphorylated proteins were analyzed by SDS-PAGE, and the gels were imaged using a phosphorimaging system (Fuji BAS1000). HPLC Analysis of Phosphopeptides—For HPLC analysis of the phosphopeptides, the phosphorylated protein was initially digested with proteases (trypsin or Glu-C). The phosphorylated protein was treated with 1% SDS and 10 mm dithiothreitol and boiled for 3 min. The protein was additionally treated with 4-vinylpyridine (0.5%) for 30 min at room temperature. SDS-PAGE was performed using 4–12% bis-Tris gels (Novex, Invitrogen) and MES running buffer. The band corresponding to PITPα was excised with a clean, sharp scalpel blade and placed into a clean siliconized tube (Sigma). The gel piece was washed sequentially with (i) water, (ii) 50% acetonitrile, (iii) 100 mm ammonium bicarbonate, and (iv) 50% acetonitrile, 100 mm ammonium bicarbonate each for 15 min. The piece was then crushed using an Eppendorf homogenizer. The resulting crushed gel pieces were incubated with 100% acetonitrile for a further 15 min and then dried under vacuum. After drying, 200 μl of Digest buffer (50 mm ammonium bicarbonate, 0.025% Zwittergent 3–16) and 2–4 μg of sequencing grade protease (trypsin or Glu-C) were added and incubated at 30 °C for 30 min. Proteases were solubilized in 1 mm HCl at 1 mg/ml. More Digest buffer was then added to completely cover the gel pieces, and the mixture was left at 30 °C for 16 h. Following overnight digestion, the supernatant was removed from the gel pieces and filtered using an Ultra-MC filter unit (Millipore). The total volume of the filtrate was determined, and an equal volume of 0.2% trifluoroacetic acid was added. The peptides were then run on a C18 Vydac column (catalog no. 218TP54). The column was run at 0.8 ml/min using water, 0.1% trifluoroacetic acid (Pump A) and 100% acetonitrile, 0.1% trifluoroacetic acid (Pump B). Gradient conditions were 0–90 min, 0–30% B; 90–110 min, 30–50% B; 110–120 min, 50–100% B; 120–130 min, 100% B; and 130–140 min, 100–0% B. Fractions (0.5 min/0.4 ml) were collected at 0–120 min. Radioactivity was determined using Cerenkov counting in a Packard liquid scintillation counter. Mutagenesis and Purification of Recombinant PITPα Proteins in Escherichia coli—Human PITPα was expressed using pET14b vector (Novagen). Mutagenesis was performed using PCR with the following mutagenesis primers: T59E, 5′-GGCCAGTACGAACACAAGATC-3′, 3′-GATCTTGTGTTCGTACTGGCC-5′; S166E, 5′-GCAAAATTTAAAGAAATCAAAACAGGCC-3′, 3′-GGCCTGTTTTGATTTCTTTAAATTTTGC-5′; T169E, 5′-TAAATCTATCAAAGAAGGCCGAGGACC-3′, 3′-GTCCTCGGCCTTCTTTGATAGATTTA-5′; T198E, 5′-CAAACTGGTGGAAGTCAAGTTCAAG-3′, 3′-CTTGAACTTGACTTCCACCAGTTTG-5′; T251E, 5′-GGAAGAAGAGGAAAAGAGACAGC-3′, 3′-GCTGTCTCTTTTCCTCTTCTTCC-5′; T59A, 5′-GGCCAGTACGCACACAAGATC-3′, 3′-GATCTTGTGTGCGTACTGGCC-5′; S166A, 5′-GCAAAATTTAAAGCAATCAAAACAGGCC-3′, 3′-GCCTGTTTTGATTGCTTTAAATTTTGC-5′. Primers for the 5′ and 3′ ends (5′-CTCCGACCATATGGTGCTGCTCAAGGAGTATCG-3′ and 5′-CCGGGATCCTAGTCATCTGCTGTCATTCC-3′) were used to ligate the mutated sequences into pET14b vector with NdeI and BamHI. Proteins were expressed in BL21(DE3)pLysS and purified using nitrilotriacetic acid-agarose (Invitrogen) as described previously (32Hara S. Swigart P. Jones D. Cockcroft S. J. Biol. Chem. 1997; 272: 14909-14913Google Scholar). The His-tagged proteins were desalted to PIPES buffer (20 mm PIPES, 137 mm NaCl, 3 mm KCl, pH 6.8), concentrated, and analyzed by SDS-PAGE. When required, the His tag was removed following cleavage with 1 unit of thrombin/1 mg of PITPα proteins (16 h at 25 °C) followed by size exclusion chromatography. Native PAGE—Thrombin-cleaved wild-type and T59A (10 μg) proteins were resolved by native PAGE. Novex Tris/glycine native gels (10–20%), Tris/glycine running buffer, and sample buffer were obtained from Invitrogen. Gels were run at a constant 125 V for 5 h at room temperature. Proteins were additionally examined by SDS-PAGE to check for purity. Reconstitution of G-protein-stimulated Phospholipase C Activity in Permeabilized HL60 Cells—The reconstitution of PLC activity was measured for each mutant using cytosol-depleted HL60 cells (8Cunningham E. Thomas G.M. Ball A. Hiles I. Cockcroft S. Curr. Biol. 1995; 5: 775-783Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). In brief, [3H]inositol-labeled HL60 cells were permeabilized with streptolysin O to remove endogenous PITPs, washed, and stimulated with GTPγS (10 μm) in the presence of Ca2+ (1 μm) and 1 mm MgATP. After incubation for 20 min at 37 °C, the production of 3H-labeled inositol phosphates was measured as an indicator of PLC activity. Transfer and Binding of PI and PC—Transfer of PI by PITPα in vitro was measured by monitoring the transfer of [3H]inositol-labeled PI from microsomes to non-labeled liposomes as described previously (28Thomas G.M. Cunningham E. Fensome A. Ball A. Totty N.F. Tro" @default.
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• W2113024008 title "Phosphorylation of a Distinct Structural Form of Phosphatidylinositol Transfer Protein α at Ser166 by Protein Kinase C Disrupts Receptor-mediated Phospholipase C Signaling by Inhibiting Delivery of Phosphatidylinositol to Membranes" @default.
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