Purification and Characterization of Gβγ-responsive Phosphoinositide 3-Kinases from Pig Platelet Cytosol
1997; Elsevier BV; Volume: 272; Issue: 22 Linguagem: Inglês
10.1074/jbc.272.22.14193
ISSN1083-351X
Autores Tópico(s)Cell Adhesion Molecules Research
ResumoA G-protein βγ subunit (Gβγ)-responsive phosphoinositide 3-kinase (PI 3-kinase) was purified approximately 5000-fold from pig platelet cytosol. The enzyme was purified by polyethylene glycol precipitation of the cytosol followed by column chromatography on Q-Sepharose fast flow, gel filtration, heparin-Sepharose, and hydroxyapatite. The major Gβγ-responsive PI 3-kinase is distinct from p85 containing PI 3-kinase as the activities can be distinguished chromatographically and immunologically and is related to p110γ as it cross-reacts with anti-p110γ-specific antibodies. The p110γ-related PI 3-kinase cannot be activated by G-protein αi/o subunits, and it has an apparent native molecular mass of 210 kDa. The p110γ-related PI 3-kinase phosphorylates phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The apparent Km values for ATP were found to be 25 μm with PtdIns, 44 μm with PtdIns4P, and 37 μm with PtdIns(4,5)P2 as the substrate. Gβγ subunits did not alter the Km of the enzyme for ATP; however,Vmax increased 2-fold with PtdIns as substrate, 3.5-fold with PtdIns4P, and 10-fold with PtdIns(4,5)P2. Under basal conditions the apparent Km values for lipid substrates were 64, 10, and 15 μm for PtdIns, PtdIns4P, and PtdIns(4,5)P2, respectively. In the presence of Gβγ subunits the dependence of PI 3-kinase activity on the concentrations of lipid substrates became complex with the highest level of stimulation occurring at high substrate concentration, suggesting that the binding of Gβγ and lipid substrate (particularly PtdIns(4,5)P2) may be mutually cooperative. Wortmannin and LY294002 inhibit the Gβγ-responsive PI 3-kinase activity with IC50 values of 10 nm and 2 μm, respectively. Unlike the p85 containing PI 3-kinase in platelets, the p110γ-related PI 3-kinase is not associated with a PtdIns(3,4,5)P3 specific 5-phosphatase.The p85-associated PI 3-kinase was not activated by Gβγ alone but could be synergistically activated by Gβγ and phosphotyrosyl platelet-derived growth factor receptor peptides. This may represent a form of coincidence detection through which the effects of tyrosine kinase and G-protein-linked receptors might be coordinated. A G-protein βγ subunit (Gβγ)-responsive phosphoinositide 3-kinase (PI 3-kinase) was purified approximately 5000-fold from pig platelet cytosol. The enzyme was purified by polyethylene glycol precipitation of the cytosol followed by column chromatography on Q-Sepharose fast flow, gel filtration, heparin-Sepharose, and hydroxyapatite. The major Gβγ-responsive PI 3-kinase is distinct from p85 containing PI 3-kinase as the activities can be distinguished chromatographically and immunologically and is related to p110γ as it cross-reacts with anti-p110γ-specific antibodies. The p110γ-related PI 3-kinase cannot be activated by G-protein αi/o subunits, and it has an apparent native molecular mass of 210 kDa. The p110γ-related PI 3-kinase phosphorylates phosphatidylinositol (PtdIns), phosphatidylinositol 4-phosphate (PtdIns4P), and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). The apparent Km values for ATP were found to be 25 μm with PtdIns, 44 μm with PtdIns4P, and 37 μm with PtdIns(4,5)P2 as the substrate. Gβγ subunits did not alter the Km of the enzyme for ATP; however,Vmax increased 2-fold with PtdIns as substrate, 3.5-fold with PtdIns4P, and 10-fold with PtdIns(4,5)P2. Under basal conditions the apparent Km values for lipid substrates were 64, 10, and 15 μm for PtdIns, PtdIns4P, and PtdIns(4,5)P2, respectively. In the presence of Gβγ subunits the dependence of PI 3-kinase activity on the concentrations of lipid substrates became complex with the highest level of stimulation occurring at high substrate concentration, suggesting that the binding of Gβγ and lipid substrate (particularly PtdIns(4,5)P2) may be mutually cooperative. Wortmannin and LY294002 inhibit the Gβγ-responsive PI 3-kinase activity with IC50 values of 10 nm and 2 μm, respectively. Unlike the p85 containing PI 3-kinase in platelets, the p110γ-related PI 3-kinase is not associated with a PtdIns(3,4,5)P3 specific 5-phosphatase. The p85-associated PI 3-kinase was not activated by Gβγ alone but could be synergistically activated by Gβγ and phosphotyrosyl platelet-derived growth factor receptor peptides. This may represent a form of coincidence detection through which the effects of tyrosine kinase and G-protein-linked receptors might be coordinated. Phosphoinositide 3-kinases (PI 3-kinases, 1The abbreviations used are:PI 3-kinasephosphoinositide 3-kinaseDTTdithiothreitolG-proteinguanine-nucleotide binding regulatory proteinGTPγSguanosine 5′-(γ-thio)triphosphatePtdInsphosphatidylinositolPtdIns4Pphosphatidylinositol 4-phosphatePtdIns(4,5)P2phosphatidylinositol 4,5-bisphosphatePSphosphatidylserinePEGpolyethylene glycolPAGEpolyacrylamide gel electrophoresisPHpleckstrin homologyPIphosphatidylinositolPLCphospholipase CPDGFplatelet-derived growth factor EC 2.7.1.137) phosphorylate the D-3 position of the inositol ring in inositol phospholipids and were originally identified through their association with viral oncoproteins and activated tyrosine kinases (1Whitman M. Downes C.P. Keller M. Keller T. Cantley L. Nature. 1988; 332: 644-646Crossref PubMed Scopus (735) Google Scholar, 2Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2185) Google Scholar). Receptor-regulated forms of PI 3-kinase appear to prefer to phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in vivo (3Stephens L.R. Hughes K.T. Irvine R.F. Nature. 1991; 351: 33-39Crossref PubMed Scopus (386) Google Scholar, 4Hawkins P.T. Jackson T.R. Stephens L.R. Nature. 1992; 358: 157-159Crossref PubMed Scopus (198) Google Scholar, 5Carter A.N. Huang R. Sorisky A. Downes C.P. Rittenhouse S.E. Biochem. J. 1994; 301: 415-420Crossref PubMed Scopus (42) Google Scholar). The product of this reaction, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3), is a candidate second messenger that regulates a variety of cellular responses to growth factors perhaps through the activation of serine/threonine protein kinases such as Akt/PKB and/or certain protein kinase C isoforms and small GTP-binding proteins such as Rac 1 (6Franke T.F. Yang S., II. Chan T.O. Ketaki D. Kazlauskas A. Morrison D.K. Kaplan D.R. Tsichlis P.N. Cell. 1995; 81: 727-736Abstract Full Text PDF PubMed Scopus (1821) Google Scholar, 7Singh S.S. Chauhan A. Brockerhoff H. Chauhan V.P.S. Biochem. Biophys. Res. Commun. 1993; 195: 104-112Crossref PubMed Scopus (68) Google Scholar, 8Nakanishi H. Brewer K.A. Exton J.H. J. Biol. Chem. 1993; 268: 13-16Abstract Full Text PDF PubMed Google Scholar, 9Toker A. Meyer M. Reddy K.K. Falck J.R. Aneja R. Aneja S. Parra A. Burns D.J. Ballas L.M. Cantley L.C. J. Biol. Chem. 1994; 269: 32358-32367Abstract Full Text PDF PubMed Google Scholar, 10Hawkins P.T. Eguinoa A. Qiu R.-G. Stokoe D. Cooke F.T. Walters R. Wennstrom S. Claesson-Welsh L. Evans T. Symons M. Stephens L. Curr. Biol. 1995; 5: 393-403Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). phosphoinositide 3-kinase dithiothreitol guanine-nucleotide binding regulatory protein guanosine 5′-(γ-thio)triphosphate phosphatidylinositol phosphatidylinositol 4-phosphate phosphatidylinositol 4,5-bisphosphate phosphatidylserine polyethylene glycol polyacrylamide gel electrophoresis pleckstrin homology phosphatidylinositol phospholipase C platelet-derived growth factor The first form of PI 3-kinase to be purified and cloned was identified as a heterodimer composed of a 110-kDa catalytic subunit with a tightly bound regulatory subunit of 85 kDa (11Otsu M. Hiles I. Gout I. Fry M.J. Ruiz-Larrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.W. Cell. 1991; 65: 91-104Abstract Full Text PDF PubMed Scopus (541) Google Scholar, 12Escobedo I.A. Navankasatlusas S. Kavanaugh W.M. Milfay D. Fried V.A. Williams L.T. Cell. 1991; 65: 75-82Abstract Full Text PDF PubMed Scopus (374) Google Scholar, 13Skolnik E.Y. Margolis B. Mohammadi M. Lowenstein E. Fischer R. Drepps A. Ullrich A. Schlessinger J. Cell. 1991; 65: 83-90Abstract Full Text PDF PubMed Scopus (437) Google Scholar, 14Hiles I.D. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz-Larrea F. Thompson A. Totty N. Hsuan J. Courtneidge S.A. Parker P.J. Waterfield M.W. Cell. 1992; 70: 419-429Abstract Full Text PDF PubMed Scopus (540) Google Scholar). The p85 subunit possesses a number of regions with homology to recognized signaling proteins. These include a Bcr homology domain, a Src homology (SH) 3, and two SH2 domains and two proline-rich regions. Binding of the SH2 domains to phosphotyrosine residues within the sequence context, YMX M, which occurs in a wide range of activated growth factor receptors and adaptor proteins, causes the translocation and activation of the catalytic subunit (2Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2185) Google Scholar, 15Fantl W. Escobedo J.A. Martin G.A. Turck C.W. del Rosario M. McCormick F. Williams L.T. Cell. 1992; 69: 413-423Abstract Full Text PDF PubMed Scopus (473) Google Scholar, 16Kashishian A. Kazlauskas A. Cooper J.A. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (201) Google Scholar). More recently, a number of distinct p85 and p110 subunit isoforms have been identified (11Otsu M. Hiles I. Gout I. Fry M.J. Ruiz-Larrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.W. Cell. 1991; 65: 91-104Abstract Full Text PDF PubMed Scopus (541) Google Scholar, 14Hiles I.D. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz-Larrea F. Thompson A. Totty N. Hsuan J. Courtneidge S.A. Parker P.J. Waterfield M.W. Cell. 1992; 70: 419-429Abstract Full Text PDF PubMed Scopus (540) Google Scholar, 17Hu P. Mondino A. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1993; 13: 7677-7688Crossref PubMed Scopus (235) Google Scholar), but the functional significance of this heterogeneity is not yet clear (18Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar). Heterotrimeric G-protein-regulated forms of PI 3-kinase have also been identified following the observations that activation of G-protein-coupled receptors in neutrophils and platelets caused a rapid accumulation of PtdIns(3,4,5)P3 (19Traynor-Kaplan A.E. Thompson B.L. Harris A.L. Taylor P. Omann G.M. Sklar L.A. Nature. 1989; 334: 353-356Crossref Scopus (208) Google Scholar, 20Kucera G.L. Rittenhouse S.E. J. Biol. Chem. 1990; 265: 5345-5348Abstract Full Text PDF PubMed Google Scholar, 21Sorisky A. King W.G. Rittenhouse S.E. Biochem. J. 1992; 286: 581-584Crossref PubMed Scopus (48) Google Scholar). Stephens et al. (22Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (519) Google Scholar) partially purified a G-protein βγ subunit (Gβγ)-responsive PI 3-kinase from a myeloid cell line (U937). This enzyme is immunologically and biochemically distinct from a growth factor-regulated, p85-containing PI 3-kinase present in the same cells. Using degenerate oligonucleotide primers based on conserved regions of known PI kinases, Stoyanov et al. (23Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnburg B. Gierschik P. Seedorf K. Hsuan J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (639) Google Scholar) isolated a p110 homologue, designated p110γ, from a U937 cell cDNA library. Recombinant p110γ has a predicted molecular mass of 120 kDa and can be activated in vitro by both Gβγ and the α subunits of transducin and Gi. p110γ differs from the α and β isoforms mainly by its lack of the recognized p85 binding site which is replaced by a region which has been proposed to resemble a pleckstrin homology (PH) domain (23Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nurnburg B. Gierschik P. Seedorf K. Hsuan J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (639) Google Scholar). A Gβγ-responsive PI 3-kinase has also been reported to be present in platelet cytosol (24Thomason P.A. James S.R. Casey P.J. Downes C.P. J. Biol. Chem. 1994; 269: 16525-16528Abstract Full Text PDF PubMed Google Scholar). Because this activity associated with a PDGF receptor phosphotyrosyl peptide and was immunoprecipitated with a monoclonal antibody raised against the p85 subunit of PI 3-kinase, it was thought to possess a p85-related subunit. However, Zhang et al. (25Zhang J. Zhang J. Benovic J.L. Sugai M. Wetzker R. Gout I. Rittenhouse S.E. J. Biol. Chem. 1995; 270: 6589-6594Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) reported a Gβγ-stimulated PI 3-kinase in platelets that was not recognized by p85-directed antibodies. The latter study established that this platelet Gβγ-stimulated enzyme was immunologically related to p110γ. The substrate specificities of identified PI 3-kinases vary substantially (11Otsu M. Hiles I. Gout I. Fry M.J. Ruiz-Larrea F. Panayotou G. Thompson A. Dhand R. Hsuan J. Totty N. Smith A.D. Morgan S.J. Courtneidge S.A. Parker P.J. Waterfield M.W. Cell. 1991; 65: 91-104Abstract Full Text PDF PubMed Scopus (541) Google Scholar, 14Hiles I.D. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz-Larrea F. Thompson A. Totty N. Hsuan J. Courtneidge S.A. Parker P.J. Waterfield M.W. Cell. 1992; 70: 419-429Abstract Full Text PDF PubMed Scopus (540) Google Scholar, 22Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (519) Google Scholar, 24Thomason P.A. James S.R. Casey P.J. Downes C.P. J. Biol. Chem. 1994; 269: 16525-16528Abstract Full Text PDF PubMed Google Scholar, 26Stephens L. Cooke F.T. Walter R. Jackson T. Volinia S. Gout I. Waterfield M.D. Hawkins P.T. Curr. Biol. 1994; 4: 203-214Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 27Macdougall L.K. Domin J. Walterfield M.D. Curr. Biol. 1995; 5: 1404-1415Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 28Volinia S. Dhand R. Vanhaesebroeck B. Macdougall L.K. Stein R. Zvelebil M.J. Domin J. Panayotou C. Waterfield M.D. EMBO J. 1995; 14: 3339-3348Crossref PubMed Scopus (307) Google Scholar). To define the molecular characteristics and properties of Gβγ-stimulated PI 3-kinases in platelets more clearly, we have now partially purified the major form of this enzyme from pig platelets. This enzyme is a p110γ-related PI 3-kinase that is distinct from p85-associated species and that phosphorylates all three potential phosphoinositide substrates with a marked preference for PtdIns(4,5)P2 which is further enhanced by Gβγ. As the results appeared to contradict previous detection of a Gβγ-responsive p85-containing PI 3-kinase in human platelets (24Thomason P.A. James S.R. Casey P.J. Downes C.P. J. Biol. Chem. 1994; 269: 16525-16528Abstract Full Text PDF PubMed Google Scholar), we also studied the p85-containing PI 3-kinase in pig platelets. We found that this enzyme can be activated by Gβγ in a manner that is largely dependent upon the presence of a tyrosine-phosphorylated PDGF receptor peptide. Polyvinylidene difluoride membrane, PtdIns, phosphatidyl-l-serine, and protein G-Sepharose were purchased from Sigma; PtdIns(4)P and PtdIns(4,5)P2 were prepared as described (38James S.R. Demel R.A. Downes C.P. Biochem. J. 1994; 298: 499-507Crossref PubMed Scopus (31) Google Scholar). [γ-32P]ATP (3000Ci/mmol) and enhanced chemiluminescence were purchased from Amersham Corp. Partisphere SAX high performance liquid chromatography columns were from Whatman; Superose 12 HR 10/30, Mono Q, Q-Sepharose fast flow, heparin-Sepharose CL-6B and PD-10 column were from Pharmacia Biotech Inc. Centricon-30 was from Amicon. GTPγS was from Boehringer Mannheim; mouse monoclonal antibodies to PI 3-kinase p85 were from Upstate Biotechnology Inc. Genapol C-100 detergent was from CAlbiochem; Gαi/owas kindly provided by Dr. P. Casey (Duke University, North Carolina). Phosphotyrosyl peptide based on the sequence of PDGF receptor was provided by Dr. S. Cartlidge, Zenaca Pharmaceutical. p110γ anti-peptide antibodies were provided by Dr. R. Wetzker (Friedrich-Schiller-Universitat Jena). The major G-protein βγ subunits were purified from cholate extracts of bovine brain membranes as described by Sternweis and Robishaw (39Sternweis P.C. Robishaw J.D. J. Biol. Chem. 1984; 259: 13806-13813Abstract Full Text PDF PubMed Google Scholar). The Gβγ subunits were stored in 20 mm Tris, 1 mm EDTA, 0.1% Genaple C-100 and were more than 95% pure as determined by SDS-PAGE. The Gβγ preparation was flash-frozen in 10-μl aliquots and stored at −80 °C until use. Platelet cytosol was derived from 12 liters of freshly drawn pig blood. The detailed procedure for preparation of platelet cytosol was essentially the same as described (24Thomason P.A. James S.R. Casey P.J. Downes C.P. J. Biol. Chem. 1994; 269: 16525-16528Abstract Full Text PDF PubMed Google Scholar). Platelets were sonicated in 120 ml of lysis buffer (10 mm HEPES, pH 7.4, 1 mm EGTA, 0.2 mm EDTA, 3 mmMgCl2, 10 mg/ml each of antipain and pepstatin, 1 mm each of DTT, sodium orthovanadate, phenylmethylsulfonyl fluoride, and benzamidine). The platelet lysate was centrifuged at 35,000 rpm for 1 h, and the resulting supernatant (platelet cytosol, 1049 mg of protein) was kept. Gβγ-responsive PI 3-kinase was precipitated by 5–15% PEG in buffer A (20 mm HEPES, pH 7.4, 0.2 mm EDTA, 3 mm MgCl2, 10 mg/ml each of antipain and pepstatin, 1 mm each of DTT, sodium orthovanadate, phenylmethylsulfonyl fluoride, and benzamidine). The pellet was resuspended in 36 ml of buffer A. The PEG sample (36 mg) was loaded onto a Q-Sepharose Fast Flow column (150 × 15 mm), pre-equilibrated with buffer A, and eluted with a gradient of 0–0.5m NaCl (150 ml). Gβγ-responsive PI 3-kinase fractions (12 mg) were pooled and loaded onto a gel filtration column (Sepharose CL-4B, 100 × 2.6 cm) pre-equilibrated with buffer B (buffer A plus 100 mm NaCl and 10% sucrose). The Gβγ-responsive PI 3-kinase activity fractions (1.35 mg protein) were pooled and loaded onto a heparin-Sepharose column (50 × 10 mm) pre-equilibrated with buffer B. The column was washed with 30 ml of buffer B and eluted with a linear gradient of 100–500 mm NaCl (60 ml). Fractions (0.36 mg) containing Gβγ-responsive PI 3-kinase were pooled and concentrated to 6 ml with Prepcentricon 30 (Amicon, Inc. Beverly, MA). Half of the sample was loaded onto a hydroxyapatite column (100 × 10) pre-equilibrated with buffer C (20 mm K2HPO4, pH 7.0, 5 mmDTT, 0.1 mm each of phenylmethylsulfonyl fluoride and benzamidine); protein was eluted with 20–750 mmK2HPO4 during which the Gβγ-responsive PI 3-kinase was separated from the tyrosine kinase-regulated PI 3-kinase. Separation of the Gβγ-responsive PI 3-kinase from the tyrosine kinase-regulated PI 3-kinase can also be achieved by incubating half of the concentrated enzyme with 2 ml of protein G-Sepharose pre-coupled with anti-p85 antibodies overnight at 4 °C with gentle agitation. The Gβγ-responsive PI 3-kinase prepared by either method did not contain any p85 protein nor any other detectable lipid kinase activity. Generally the enzyme activity was measured by adopting the following assay procedure. 10 μl of platelet cytosol or column fractions were mixed with 30 μl of lipid vesicles, which had been premixed with Gβγ or their vehicle for 10 min on ice. 10 μl of MgATP was added to start the reaction. The enzyme reaction was terminated after incubating at 37 °C for 5 min by adding 200 μl of 1 m HCl. To prepare lipid vesicles, equimolar amounts of PS and substrate lipid (PtdIns, PtdIns4P, or PtdIns(4,5)P2) were dried onto a film under vacuum and probe-sonicated (3 × 15 s with 1 min on ice between sonication, at setting 20–30 on a Jencons Ultrasonic Processor) into kinase assay buffer (40 mm HEPES, pH 7.4, 1 mmEGTA, 1 mm DTT, 50 mm NaCl, 4 mmMgCl2). The standard assay contained 100 μmPtdIns(4,5)P2 and PS, 10–100 μm ATP (10 μm with purified fraction and 100 μm with crude extract), 1 μm Gβγ or its vehicles and 10 μCi of [γ32P]ATP. Lipid extraction and analysis was performed as described (24Thomason P.A. James S.R. Casey P.J. Downes C.P. J. Biol. Chem. 1994; 269: 16525-16528Abstract Full Text PDF PubMed Google Scholar). The products of the PI 3-kinase reactions were identified by deacylation and separation of their glycerol derivatives by high performance liquid chromatography and compared with deacylated 3H-labeled standards. Note that assays were done under first order conditions with respect to ATP as substrate (to optimize assay of radioactive product) ensuring that no more than 10% of ATP was consumed during a reaction. For assays requiring activated Gα-proteins, Gα-proteins were incubated for 1 h on ice in the presence of 100 μmGTPγS and 5 mm MgCl2, mixed with PtdIns-containing lipid vesicles, and incubated again for 10 min on ice, before adding to the assay. Samples of column eluate (for detection of p85 subunit of PI 3-kinase) or purified Gβγ-responsive PI 3-kinase (for p110γ) were mixed with 4 × SDS sample buffer, boiled for 5 min, and resolved by SDS-PAGE with 7.5% acrylamide in the separating gel. Proteins were then transferred to polyvinylidene difluoride membranes using a dry-blotting device (Bio-Rad). Western blots were performed as described (22Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (519) Google Scholar) using either anti-p85 antibody (1:1000 dilution) or p110γ antibodies (1:200) followed by horseradish peroxidase-conjugated secondary antibodies (1:2000 dilution). Blots were then developed using enhanced chemiluminescence according to the manufacturer's instructions. A Gβγ-responsive PI 3-kinase was purified approximately 5000-fold, with an overall yield of 30%, from porcine platelet cytosol using PEG precipitation, Q-Sepharose, gel filtration, heparin-Sepharose, and hydroxyapatite. A typical purification is summarized in Table I. PEG precipitation resulted in a 30-fold enrichment with 100% recovery of Gβγ-responsive activity. Elution of this sample from Q-Sepharose using a continuous salt gradient revealed two distinct peaks of Gβγ-responsive PI 3-kinase (Fig. 1 A). The earlier eluting, minor peak of activity was inconsistently observed in a number of purifications from different batches of platelets and was not studied further. Analysis of fractions eluting from the Q-Sepharose column by Western blotting with an anti-p85 monoclonal antibody revealed that the second peak of Gβγ-responsive PI 3-kinase co-eluted with p85 immunoreactivity (Fig. 1 B). p85 and Gβγ-responsive activity continued to co-migrate through gel filtration and heparin-Sepharose (data not shown). However, separation of p85 and the Gβγ-responsive PI 3-kinase was achieved through hydroxyapatite eluted with a linear gradient of K2HPO4/KH2PO4 as shown in Fig. 2, A and B. Separation could also be achieved by immunodepletion of p85 using protein G-Sepharose that had been pre-coupled with anti-p85 antibodies (Fig.3 A). The Gβγ-responsive PI 3-kinase is related to p110γ as it can be recognized by an anti-p110γ anti-peptide antibody (Fig. 3 B).Table IPurification of Gβγ-responsive PI 3-kinaseStepTotal Gβγ stimulatable activityTotal proteinSpecific activityYieldPurificationnmol PIP3 · min−1mgnmol PIP3 · mg−1 · min−1-foldCrude platelet cytosol4.5010490.0042100PHG 80004.60360.1310230Q-Sepharose3.83120.328576Gel filtration3.601.352.6780635Heparin-Sepharose3.020.368.40672000Hydroxyapatite1.350.0621.00305000The purification began with 1049 mg of platelet cytosol protein from 12 liters of blood. Columns were run as described under "Experimental Procedures." The Gβγ-stimulatable PI 3-kinase activity was defined as the activity in the presence of Gβγ minus that in the absence of Gβγ in the assay and is expressed as nmol of PtdInsP3 formed per min. Open table in a new tab Figure 2A–B, purification of Gβγ-responsive PI 3-kinase on hydroxyapatite. A, the heparin-Sepharose-eluted sample (0.18 mg) was loaded onto a hydroxyapatite column. Protein was eluted with a gradient of K2HPO4. Aliquots of individual fractions were immediately assayed for PI(4,5)P2 3-kinase activity in the presence and absence of 1 μm Gβγ. ▪, stimulated; □, basal; ⎻, K2HPO4. B, aliquots of fractions were analyzed by SDS-PAGE and blotted with anti-p85 antibodies.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3A-B, immunoprecipitation (IP) of the p85-containing PI 3-kinase. A sample of heparin-Sepharose-purified enzyme was mixed with protein G-Sepharose that had been pre-coupled with anti-p85 antibodies. Aliquots of the heparin-Sepharose sample, supernatant, and precipitate were analyzed by SDS-PAGE and blotted with either anti-p85 antibodies (A) or blotted with anti-p110γ antibodies (B).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The purification began with 1049 mg of platelet cytosol protein from 12 liters of blood. Columns were run as described under "Experimental Procedures." The Gβγ-stimulatable PI 3-kinase activity was defined as the activity in the presence of Gβγ minus that in the absence of Gβγ in the assay and is expressed as nmol of PtdInsP3 formed per min. The native molecular mass of the Gβγ-responsive PI 3-kinase was determined using a calibrated Superose 12 size exclusion column. As shown in Fig.4, the activity eluted at a volume indicating a size of approximately 210 kDa. The enzyme was not pure at that stage as revealed by silver-stained SDS-PAGE gel of active fractions (not shown). This indicated that the enzyme is a very minor protein in platelet cytosol. The substrate specificity and kinetic characteristics of the enzyme were consistent between several different preparations. As the enzyme after hydroxyapatite cannot survive freezing and thawing, activity purified through heparin-Sepharose and immunodepleted to remove p85 was used for most experiments. Such preparations were purified approximately 2000-fold with respect to platelet cytosol and contained no detectable PtdIns 4-kinase, PtdIns4P 5-kinase, or PLC (EC 3.1.4.3) activities. PtdIns, PtdIns4P, and PtdIns(4,5)P2 were all phosphorylated at the 3-position (see "Experimental Procedures"), and the phosphorylations of PtdIns and PtdIns4P were inhibited by the presence of PtdIns(4, 5)P2 as described previously (29Abrams C.S. Zhang J. Downes C.P. Tang X. Zhao W. Rittenhouse S.E. J. Biol. Chem. 1996; 271: 25192-25197Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) indicating that a single enzyme activity accounts for the phosphorylation of all three substrates. Anti-p85 and anti-p110 antibodies were recently shown to co-immunoprecipitate with a PtdIns(3,4,5)P3-specific 5-phosphatase which was presumed to be present in a complex with this form of PI 3-kinase (30Jackson S.P. Schoenwaelder S.M. Matzaris M. Brown S. Mitchell C.A. EMBO J. 1995; 14: 4490-4500Crossref PubMed Scopus (74) Google Scholar). Analysis of the partially purified Gβγ-sensitive PI 3-kinase using32P-labeled PtdIns(3,4,5)P3 as substrate or by monitoring the ratio of PtdIns(3,4,5)P3/PtdIns(3,4)P2 in PI 3-kinase assays failed to detect any 5-phosphatase in such preparations (data not shown). Km values were determined under basal conditions for ATP and all three lipid substrates. Similar Kmvalues for ATP of 25, 44, and 37 μm were determined when PtdIns, PtdIns4P, or PtdIns(4,5)P2, respectively, were used as substrates (Fig. 5 A and Fig.6 A). Km values for the lipid substrates were determined using 10 μm ATP and maintaining a constant mole fraction of 1:1 (substrate/PS). 10 μm ATP (i.e. somewhat less than theKm value) was used to estimate the sensitivity of the assays. However, since less than 5% ATP was consumed during incubation, the assays were linear under the conditions determined. The reactions approximated to Michaelis-Menten kinetics withKm values of 64, 10, and 15 μm for PtdIns, PtdIns4P, and PtdIns(4,5)P2, respectively (Fig.5 B and Fig. 6 B). Similar values were obtained using either Lineweaver-Burk or Wolfe plots.Figure 6Double-reciprocal plots of kinetic data of Gβγ-responsive PI 3-kinase in the absence of Gβγ. A, various concentrations of PI/PS, PI-4P/PS, or PI(4,5)P2/PS.B, various concentrations of [γ-32P]ATP. □, PI; ⋄, PI4P; ○, PI(4,5)P2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The effects of increasing concentrations of Gβγ subunits were examined at an ATP concentration of 10 μm with lipid substrate concentrations of 100 μm. Interestingly the EC50 values differed according to the lipid substrate as noted previously for the human platelet cytosol activity (24Thomason P.A. James S.R. Casey P.J. Downes C.P. J. Biol. Chem. 1994; 269: 16525-16528Abstract Full Text PDF PubMed Google Scholar). When PtdIns(4,5)P2
Referência(s)