Human Phosphoinositide 3-Kinase C2β, the Role of Calcium and the C2 Domain in Enzyme Activity
1998; Elsevier BV; Volume: 273; Issue: 49 Linguagem: Inglês
10.1074/jbc.273.49.33082
ISSN1083-351X
AutoresAlexandre Arcaro, Stefano Volinia, Marketa Zvelebil, Robert C. Stein, Sandra J. Watton, Meredith J. Layton, Ivan Gout, Khatereh Ahmadi, Julian Downward, Michael D. Waterfield,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoThe cDNA for a human Class II phosphoinositide 3-kinase (PI 3-kinase C2β) with a C2 domain was cloned from a U937 monocyte cDNA library and the enzyme expressed in mammalian and insect cells. Like other Class II PI 3-kinasesin vitro, PI 3-kinase C2β utilizes phosphatidylinositol (PI) and PI 4-monophosphate but not PI 4,5-biphosphate as substrates in the presence of Mg2+. Remarkably, and unlike other PI 3-kinases, the enzyme can use either Mg-ATP or Ca-ATP to generate PI 3-monophosphate. PI 3-kinase C2β, like the Class I PI 3-kinases, but unlike PI 3-kinase C2α, is sensitive to low nanomolar levels of the inhibitor wortmannin. The enzyme is not regulated by the small GTP-binding protein Ras. The C2 domain of the enzyme bound anionic phospholipids such as PI and phosphatidylserine in vitro, but did not co-operatively bind Ca2+ and phospholipids. Deletion of the C2 domain increased the lipid kinase activity suggesting that it functions as a negative regulator of the catalytic domain. Although presently it is not known whether PI 3-kinase C2β is regulated by Ca2+ in vivo, our results suggest a novel role for Ca2+ ions in phosphate transfer reactions. The cDNA for a human Class II phosphoinositide 3-kinase (PI 3-kinase C2β) with a C2 domain was cloned from a U937 monocyte cDNA library and the enzyme expressed in mammalian and insect cells. Like other Class II PI 3-kinasesin vitro, PI 3-kinase C2β utilizes phosphatidylinositol (PI) and PI 4-monophosphate but not PI 4,5-biphosphate as substrates in the presence of Mg2+. Remarkably, and unlike other PI 3-kinases, the enzyme can use either Mg-ATP or Ca-ATP to generate PI 3-monophosphate. PI 3-kinase C2β, like the Class I PI 3-kinases, but unlike PI 3-kinase C2α, is sensitive to low nanomolar levels of the inhibitor wortmannin. The enzyme is not regulated by the small GTP-binding protein Ras. The C2 domain of the enzyme bound anionic phospholipids such as PI and phosphatidylserine in vitro, but did not co-operatively bind Ca2+ and phospholipids. Deletion of the C2 domain increased the lipid kinase activity suggesting that it functions as a negative regulator of the catalytic domain. Although presently it is not known whether PI 3-kinase C2β is regulated by Ca2+ in vivo, our results suggest a novel role for Ca2+ ions in phosphate transfer reactions. phosphoinositide 3-kinase high pressure liquid chromatography homology region glutathione S-transferase phosphatidylcholine phosphatidylethanolamine phosphatidylinositol phosphatidylinositol 3-monophosphate phosphatidylinositol 4-monophosphate 4)P2, phosphatidylinositol 3,4-biphosphate 5)P2, phosphatidylinositol 4,5-biphosphate 4,5)P3, phosphatidylinositol 3,4,5-triphosphate phosphatidylserine polymerase chain reaction Src homology 2 and 3, respectively polyacrylamide gel electrophoresis polymerase chain reaction phosphate-buffered saline. The cell, through diverse surface receptors with unique binding specificities, can sense bound signal molecules and transduce responses that regulate its physiology. It seems clear that most cell surface receptors activate a phosphoinositide 3-kinase (PI 3-kinase)1 as part of the signal transduction cascade leading to the formation of phosphoinositides with 3′-phosphate groups (1Kapeller R. Cantley L.C. Bioessays. 1994; 16: 565-576Crossref PubMed Scopus (553) Google Scholar, 2Stephens L.R. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar). The diversity of physiological events associated with increased PI 3-kinase activity is evident from reports of enzyme activation in: processes such as cell proliferation and transformation (3Cantley 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 (2187) Google Scholar, 4Valius M. Kazlaukas A. Cell. 1993; 73: 321-334Abstract Full Text PDF PubMed Scopus (570) Google Scholar, 5Leevers S.J. Weinkove D. MacDougall L.K. Hafen E. Waterfield M.D. EMBO J. 1997; 15: 6584-6594Crossref Scopus (417) Google Scholar), events linked to insulin action, including alterations in glucose transport (6Hara K. Yonezawa K. Sakaue H. Ando A. Kotani K. Kitamura T. Kitamura Y. Ueda Y. Stephens L.R. Jackson T.R. Hawkins P.T. Dhand R. Clark A.E. Holman G.D. Waterfield M.D. Kasuga M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7415-7419Crossref PubMed Scopus (418) Google Scholar), the effects of growth factors on cell shape and motility (7Wennström S. Hawkins P.T. Cooke F. Hara K. Yonezawa K. Kasuga M. Jackson T. Claesson-Welsh L. Stephens L. Curr. Biol. 1994; 4: 385-393Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar), T cell signaling (8Exley M. Varticovski L. Peter M. Sancho J. Terhorst C. J. Biol. Chem. 1994; 269: 15140-15146Abstract Full Text PDF PubMed Google Scholar, 9Pages F. Ragueneau M. Rottapel R. Truneh A. Nunes J. Imbert J. Olive D. Nature. 1994; 369: 327-329Crossref PubMed Scopus (347) Google Scholar) and apoptosis (10Marte B.M. Downward J. Trends Biochem. Sci. 1997; 22: 355-358Abstract Full Text PDF PubMed Scopus (649) Google Scholar, 11Franke T.F. Kaplan D.R. Cantley L.C. Cell. 1997; 88: 435-437Abstract Full Text Full Text PDF PubMed Scopus (1528) Google Scholar). The activation of PI 3-kinase in this array of receptor-triggered processes suggests that 3′-phosphoinositides have a role as second messengers. At least three 3′-phosphoinositides are produced in cells: PI(3)P, PI(3,4)P2, and PI(3,4,5)P3. Receptor-triggered signals have been shown to activate PI 3-kinases and generate PI(3,4)P2 and PI(3,4,5)P3 (12Stephens L.R. Hughes K. Irvine R.F. Nature. 1991; 351: 33-39Crossref PubMed Scopus (388) Google Scholar, 13Hawkins P.T. Jackson T.R. Stephens L.R. Nature. 1992; 359: 157-159Crossref Scopus (199) Google Scholar). PI(3)P can also be detected in cells but its production is not regulated by external signals (12Stephens L.R. Hughes K. Irvine R.F. Nature. 1991; 351: 33-39Crossref PubMed Scopus (388) Google Scholar, 13Hawkins P.T. Jackson T.R. Stephens L.R. Nature. 1992; 359: 157-159Crossref Scopus (199) Google Scholar). PI(3)P and PI(3,4)P2 can also be generated from PI(3,4)P2 and PI(3,4,5)P3 through the action of phosphoinositide phosphatases which could be regulated by distinct mechanisms to those of the phosphoinositide kinases (14Woscholski R. Parker P.J. Trends Biochem. Sci. 1997; 22: 427-431Abstract Full Text PDF PubMed Scopus (71) Google Scholar). Through many studies involving the purification and molecular characterization of PI 3-kinases, a family of enzymes has been defined, which can be divided into three classes, whose members have diverse substrate specificity and distinct control mechanisms (15Zvelebil M.J. MacDougall L.K. Leevers S. Volinia S. Vanhaesebroeck B. Gout I. Panayotou G. Domin J. Stein R. Pages P. Koga H. Salim K. Linacre J. Das P. Panaretou C. Wetzker R. Waterfield M.D. Philos. Trans. R. Soc. Lond. Biol. 1996; 351: 217-223Crossref PubMed Scopus (89) Google Scholar, 16Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M.D. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (835) Google Scholar). The Class I PI 3-kinases, which can be subdivided into IA and IB, are known to be activated by receptors. Although they can phosphorylate PI, PI(4)P, and PI(4,5)P2 in vitro, these enzymes utilize mainly PI(4,5)P2 as a substrate in vivo(12Stephens L.R. Hughes K. Irvine R.F. Nature. 1991; 351: 33-39Crossref PubMed Scopus (388) Google Scholar, 13Hawkins P.T. Jackson T.R. Stephens L.R. Nature. 1992; 359: 157-159Crossref Scopus (199) Google Scholar). The Class IA enzymes are heterodimers that are recruited to, and activated by receptors linked to tyrosine kinases through their p85 subunits. At least three distinct p110 subunits that have kinase activity are associated with p85 subunits: these are p110α, p110β, and p110δ (17Hiles I.D. Otsu M. Volinia S. Fry M.J. Gout I. Dhand R. Panayotou G. Ruiz-Larrea F. Thompson A. Totty N.F. Hsuan J.J. Courtneidge S.A. Parker P.J. Waterfield M.D. Cell. 1992; 70: 419-429Abstract Full Text PDF PubMed Scopus (541) Google Scholar, 18Hu P. Mondino A. Skolnok E.Y. Schlessinger J. Mol. Cell. Biol. 1993; 13: 7677-7688Crossref PubMed Scopus (236) Google Scholar, 19Vanhaesebroeck B. Welham M.J. Kotani K. Stein R. Warne P.H. Zvelebil M.J. Higashi K. Volinia S. Downward J. Waterfield M.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4330-4335Crossref PubMed Scopus (374) Google Scholar). The p85 subunits serve as adaptors and regulators (20Otsu 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.D. Cell. 1991; 65: 91-104Abstract Full Text PDF PubMed Scopus (541) Google Scholar, 21Inukai K. Anai M. Van Breda E. Hosaka T. Katagiri H. Funaki M. Fukushima Y. Ogihara T. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1996; 271: 5317-5320Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 22Pons S. Asano T. Glasheen E. Miralpeix M. Zhang Y.T. Fisher T.L. Myers M.G. Sun X.J. White M.F. Mol. Cell. Biol. 1995; 15: 4453-4465Crossref PubMed Scopus (232) Google Scholar). The catalytic subunits can be directly regulated by Ras (23Rodriguez-Viciana P. Warne P.H. Dhand R. Vanhaesebroeck B. Gout I. Fry M.J. Waterfield M.D. Downward J. Nature. 1994; 270: 527-532Crossref Scopus (1731) Google Scholar). The Class IB PI 3-kinase, p110γ (24Stoyanov B. Volinia S. Hanck T. Rubio I. Loubtchenkov M. Malek D. Stoyanova S. Vanhaesebroeck B. Dhand R. Nümberg B. Gierschik P. Seedorf K. Hsuan J.J. Waterfield M.D. Wetzker R. Science. 1995; 269: 690-693Crossref PubMed Scopus (641) Google Scholar), is associated with a p101 adaptor, which may link the kinase to serpentine receptors by the βγ subunits of heterotrimeric G-proteins and thus mediate p110γ activation (25Stephens L.R. Eguinoa A. Erdjument-Bromage H. Lui M. Cooke F. Coadwell J. Smrcka A.S. Thelen M. Cadwaller K. Tempst P. Hawkins P.T. Cell. 1997; 89: 105-114Abstract Full Text Full Text PDF PubMed Scopus (494) Google Scholar). The Class II PI 3-kinases have a carboxyl-terminal C2 domain. The nature of any receptor-linked activation pathway for these enzymes remains unclear. Studies of the Drosophila (26MacDougall L.K. Domin J. Waterfield M.D. Curr. Biol. 1995; 5: 1404-1415Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 27Molz L. Chen Y.-W. Hirano M. Williams L.T. J. Biol. Chem. 1996; 271: 13892-13899Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), murine (27Molz L. Chen Y.-W. Hirano M. Williams L.T. J. Biol. Chem. 1996; 271: 13892-13899Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 28Virbasius J.V. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 13304-13307Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), and human enzymes (29Domin J. Pages F. Volinia S. Rittenhouse S.E. Zvelebil M.J. Stein R.C. Waterfield M.D. Biochem. J. 1997; 326: 139-147Crossref PubMed Scopus (219) Google Scholar) show that their in vitrosubstrate specificity is restricted to PI and PI(4)P, and that they cannot utilize PI(4,5)P2. The role of the C2 domain is not understood, although studies of the Drosophila enzyme suggest that it mediates calcium-independent phospholipid binding (26MacDougall L.K. Domin J. Waterfield M.D. Curr. Biol. 1995; 5: 1404-1415Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). This feature is similar to that of the C2B domain of synaptotagmin, which binds to the clathrin-AP2 complex in a Ca2+-independent manner (30Zhang J.Z. Davletov B.A. Südhof T.C. Anderson R.G.W. Cell. 1994; 78: 751-760Abstract Full Text PDF PubMed Scopus (435) Google Scholar). The synaptotagmin C2B domain can also homodimerize in a Ca2+-dependent manner (31Chapman E.R. An S. Edwardson J.M. Jahn R. J. Biol. Chem. 1996; 271: 5844-5849Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The diversity in biochemical function which can be mediated by C2 domains is evident from studies which show that C2 domains of synaptotagmin, protein kinase C, and phospholipase C can bind a variety of ligands, including Ca2+, phopholipids, inositol polyphosphates, and intracellular proteins (32Ponting C.P. Parker P. Protein Sci. 1996; 5: 162-166Crossref PubMed Scopus (155) Google Scholar, 33Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (691) Google Scholar). The third class of PI 3-kinases contains phosphatidylinositol 3-kinases that are specific for PI. Only one enzyme has yet been found in each of several species examined. In yeast, the enzyme is the product of the vesicle protein sorting mutant gene Vps34 (34Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (806) Google Scholar). Both Vps34p and its human homologue PtdIns 3-kinase (35Volinia S. Dhand R. Vanhaesebroeck B. MacDougall L.K. Stein R. Zvelebil M.J. Domin J. Panaretou C. Waterfield M.D. EMBO J. 1995; 14: 3339-3348Crossref PubMed Scopus (308) Google Scholar) associate with a serine/threonine kinase, Vps15p in yeast (36Stack J.H. Herman P.K. Schu P.V. Emr S.D. EMBO J. 1993; 12: 2195-2204Crossref PubMed Scopus (266) Google Scholar) and p150 in man (37Panaretou 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). The biochemical function of the PI(3)P generated by these enzymes has been elusive, although recent reports of alterations in PI(3,5)P2 levels in yeast and mammalian cells after osmotic shock suggest that PI(3)P may serve as a substrate for an as yet unknown PI(3)P 5-kinase (38Dove S.K. Cooke F.T. Douglas M.R. Sayers L.G. Parker P.J. Michell R.H. Nature. 1997; 390: 187-192Crossref PubMed Scopus (393) Google Scholar) and that PI(3,5)P2 may mediate vesicular trafficking. Several other proteins (Tor1 and 2, RAFT, ATM, and DNA-dependent protein kinase), which have regions homologous to the PI 3-kinase HRI kinase domain, have been described (39Hunter T. Cell. 1995; 83: 1-4Abstract Full Text PDF PubMed Scopus (261) Google Scholar). These proteins seem to be involved in cell cycle regulation and, although in some cases they have protein kinase activity, it remains unclear if they function as PI 3-kinases. In this article, we describe the cloning, expression, and enzymology of a human Class II PI 3-kinase with an amino acid sequence that is virtually identical to that of the PI 3-kinase HsC2, which was recently described by Brown et al. (40Brown R.A. Ho L.K.F. Weber-Hall S.J. Shipley J.M. Fry M.J. Biochem. Biophys. Res. Commun. 1997; 233: 537-544Crossref PubMed Scopus (61) Google Scholar). Here we show that PI 3-kinase C2β has a substrate specificity restricted to PI and PI(4)P, similar to that of the Drosophila, human, and murine Class II enzymes previously examined. The cofactor studies described here, however, show that the enzyme is unique in being able to use Ca-ATP for its lipid kinase activity in vitro. The role of the C2 domain in calcium binding, enzyme activity, and membrane association has been investigated, and the results show that the C2 domain may function mainly as a modulator of catalytic function. HEK 293 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% (v/v) heat-inactivated fetal calf serum (Life Technologies, Inc.) at 37 °C in a humidified atmosphere containing 10% CO2 and passaged every 2–3 days using Versene (Life Technologies, Inc.). Sf9 cells were grown at 27 °C in IPL-41 (Sigma) with 10% (v/v) heat-inactivated fetal calf serum supplemented with yeast extract ultrafiltrate (Sigma) and lipid concentrate (Sigma) and passaged every 3 days. Reverse transcriptase-PCR was performed on a U937 cDNA library, using degenerate primers corresponding to the conserved peptides GDDLRQD and FHIDFG, and screening was performed as described previously in Refs. 35Volinia S. Dhand R. Vanhaesebroeck B. MacDougall L.K. Stein R. Zvelebil M.J. Domin J. Panaretou C. Waterfield M.D. EMBO J. 1995; 14: 3339-3348Crossref PubMed Scopus (308) Google Scholar and 41Volinia S. Hiles I.D. Ormondroyd E. Nizetic D. Antonacci R. Rocchi M. Waterfield M.D. Genomics. 1994; 24: 472-477Crossref PubMed Scopus (98) Google Scholar. Sequencing of the cDNA clones was carried out using the Taq DyeDeoxy terminator Cycle Sequencing system (ABI) and an automated DNA sequencer (ABI 373). Rapid amplifiction of cDNA ends PCR was performed using U937 λ-ligated cDNA as a template and nested sense λ primers and PI 3-kinase C2β specific antisense primers.Taq DNA polymerase (Cetus) was used in two consecutive 50-μl PCR reactions (first PCR: ATTAACCCTCACTAAAGGG (T3 promoter) sense primer; ACTGAATTCTCAACCCACGTCCACATTCCTCAGG (+1170), antisense primer, 94 °C for 2 min denaturation followed by 30 cycles of 95 °C for 40 s, 56 °C for 15 s, 72 °C for 2 min; second PCR: TGCAGGAATTCGGCACGA (cloning site) sense primer, ACTGAATTCTCAAGTAGTCTTGGATGTCAGAGC (+971) nested antisense primer, 96 °C for 1 min followed by 25 cycles of 96 °C for 40 s, 56 °C for 15 s, 72 °C for 2 min). Approximately 500 ng of cDNA ligation mixture was used in the first PCR, then 1 μl from the first PCR reaction was amplified in the second reaction. The PCR reaction products were digested with EcoRI and ligated into pBluescript (Stratagene). Fifty clones were sequenced and oligonucleotides were designed in order to introduce anEcoRI site 5′ to the most NH2-terminal ATG codon. Reverse transcriptase-PCR using Vent DNA polymerase (New England Biolabs) was performed using U937 cDNA as a template. The resulting 5′ cDNA was digested with EcoRI and EcoRV, sequenced to check sequence fidelity and ligated in front of the remainder of the cDNA to produce the complete open reading frame. The cDNA encoding PI 3-kinase C2β was subcloned into pcDNA3 (Invitrogen) using theEcoRI and XhoI sites. NH2-terminal Glu- (MEFMPME) or Myc- (MEQKLISEEDL) epitope tags were introduced into the cDNA in-frame by PCR using Vent DNA polymerase. AnEcoRI site was added to the 5′ of the tag sequences to facilitate subcloning. The PCR products encoding the tagged NH2 termini were fused to the cDNA of PI 3-kinase C2β using a unique BstUI site at +169 and recloned into pcDNA3 using EcoRI and XhoI. The sequence of these constructs was confirmed by NH2-terminal sequencing. In order to generate a deletion of the C2 domain of PI 3-kinase C2β, the cDNA encoding the NH2-terminal Myc-tagged version of the enzyme cloned in pcDNA3 was digested at position +4419 withApaI. After dephosphorylization of the linearized DNA with calf intestinal phosphatase (Boehringer Mannheim), annealed 5′-phosphorylated oligonucleotides (sense TGAAGTACTCAATTGGGCC, antisense CAATTGAGTACTTCAGGCC) were ligated to the cDNA to introduce a TGA in-frame at codon +1474. The construct was analyzed by restriction mapping and sequencing of the 3′ end. HEK293 cells were grown to 50–60% confluence on 150-mm dishes and transfected with cDNA constructs in pcDNA3 using calcium-phosphate, exactly as described (42Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The cells were harvested 48 h after transfection and analyzed for gene expression. The cDNA construct encoding Glu-tagged PI 3-kinase C2β was subcloned into pBluescript SK usingEcoRI and XhoI sites and this construct digested with EcoRI and KpnI. The resulting cDNA insert was then subcloned into the pAcSG2 baculovirus transfer vector (PharMingen). Sf9 insect cells were co-transfected with the recombinant transfer vector encoding PI 3-kinase C2β and BaculoGold linearized baculovirus DNA (PharMingen), using Lipofectin (Life Technologies, Inc.). Recombinant baculoviruses were harvested 7 days post-transfection, amplified, and screened for induction of protein expression. Positive viruses were plaque-purified, re-amplified, and used for production of recombinant enzyme. Sf9 cells were grown to 50–60% confluency and infected with recombinant plaque-purified baculoviruses for 60 h. The cells were then harvested by centrifugation and washed once in ice-cold PBS (Life Technologies, Inc.). Recombinant PI 3-kinase C2β was purified from the Triton X-100 soluble fraction by immunoprecipitation with purified monoclonal anti-Glu-tag antibodies and Protein G-Sepharose, as described below. Cells grown on 150-mm dishes were placed on ice, washed once in ice-cold PBS (Life Technologies, Inc.), and lysed for 20 min on ice in 2 ml of lysis buffer (20 mmHEPES-NaOH (pH 7.4), 150 mm NaCl, 1% (w/v) Triton X-100, 2 mm EDTA, 10 mm sodium fluoride, 10 mm Na2HPO4, 10% (w/v) glycerol, 1 mm phenylmethylsulfonyl fluoride, 5 mmbenzamidine, 7 mm diisopropyl fluorophosphate, 1 mm N α-tosyl-l-lysine chloromethyl ketone, 20 μm leupeptin, 18 μmpepstatin, 21 μg/ml aprotinin, 2 mm dithiothreitol). The cells were then scraped from the dishes, transferred into 1.5-ml microcentrifuge tubes, and centrifuged for 20 min at 15,000 ×g at 4 °C. The supernatant was collected and incubated for 2 h at 4 °C with constant rotation with the relevant antibody. Protein A- or Protein G-Sepharose CL-4B (Pharmacia, 10 μl of beads per sample) were then added and the incubation continued for 1 h at 4 °C with constant rotation. The immunoprecipitates were washed once in lysis buffer, once in 50 mm Tris-HCl (pH 7.4), 0.5 m LiCl and once in TBS (50 mmTris-HCl (pH 7.4), 150 mm NaCl). The enzyme preparations were stored at −30 °C in TBS containing 50% glycerol and 1 mm dithiothreitol. In the case of the C2 deletion mutant, the washing and storage buffers were supplemented with 100 μm diisopropyl fluorophosphate, since the enzyme lost activity much more rapidly than the wild-type, possibly because of an increased susceptibility to proteolysis. SDS-PAGE was performed using buffer systems according to previous methods (43Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207522) Google Scholar). Polyacrylamide gels (7.5% acrylamide) were transferred onto polyvinylidene difluoride membranes (Gelman Sciences) using a semi-dry blotter (Pharmacia, 3 h at 300 mÅ). The membranes were then blocked for 1 h in PBS containing 3% (w/v) non-fat dry milk, 0.1% (w/v) PEG 20000. The relevant primary antibodies were diluted in PBS, 0.05% (w/v) Tween 20 (PBS/Tween) and incubated with the membranes for 2 h. After extensive washing in PBS/Tween, the blots were incubated for 1 h with goat anti-mouse or anti-rabbit antibodies coupled to horseradish peroxidase (Dako) at 1:2000 dilution. The membranes were then washed in PBS/Tween and the bands detected using ECL (Amersham). A cDNA fragment encoding amino acids 1 to 331 obtained by PCR was subcloned into pGEX-2T (Pharmacia) using the EcoRI 5′ of the start codon and anEcoRI site introduced by PCR at the 3′ end. Expression of this construct in Escherichia coli strain BL21/DE3 was induced by 1 mmisopropyl-1-thio-β-d-galactopyranoside. The bacteria were then harvested, snap-frozen, and lysed by sonication in ice-cold extraction buffer (EB, 10 mm TrisHCl (pH 7.4), 150 mm NaCl, 1% (w/v) Triton X-100, 5 mm EDTA, 5 mm benzamidine, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin). After centrifugation for 20 min at 15,000 × g at 4 °C, the cleared lysate was incubated with glutathione-Sepharose CL-4B (Pharmacia) for 2 h at 4 °C with constant rotation. The beads were then washed three times in EB, once in TBS, and resuspended in 50 mm Tris-HCl (pH 7.4), 2.5 mm CaCl2. Thrombin was added (4 μg/mg of recombinant protein) for 20 min at room temperature on a wheel. The supernatant containing the recombinant NH2-terminal fragment was treated withp-aminobenzamidine-agarose (Sigma), concentrated to 1 mg/ml using a Centriplus 10 concentrator (Amicon), and stored at −70 °C. The purified recombinant NH2-terminal fragment was used to immunize 2 rabbits (Eurogentec, Seraing-Belgium). The animals were injected 3 times over 35 days with 100 μg of antigen in Freund's adjuvant and the serum was collected after 42 days. The rabbits were then boosted every month and serum was collected 10 days after each injection. A cDNA fragment starting at codon 1440 and encoding the C2 domain was amplified by PCR and cloned into pGEX-2T using two EcoRI sites introduced at both the 5′ and 3′ ends during PCR. The orientation of the construct was checked by restriction digestions and sequencing. E. coli strain BL21/DE3 was transformed with the construct and protein expression induced by adding 1 mmisopropyl-1-thio-β-d-galactopyranoside. The recombinant protein was purified from the Triton X-100 soluble fraction as above, and eluted from the glutathione-Sepharose CL-4B as a GST-fusion using 100 mm Tris-HCl (pH 7.4), 150 mm NaCl, 20 mm reduced glutathione. Buffer exchange to TBS was performed on a PD-10 column (Pharmacia). The purified GST-C2 domain was concentrated to 1 mg/ml using a Centriplus 10 concentrator (Amicon) and stored at −70 °C. The same protocol was used to prepare recombinant synaptotagmin C2A and C2B domains as GST fusions. The binding of purified recombinant GST-C2 domains to phospholipid vesicles was studied using a sedimentation assay, essentially as described in Ref. 44Fukuda M. Kojima T. Mikoshiba K. J. Biol. Chem. 1996; 271: 8430-8434Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar. Phospholipids in CHCl3 (Sigma, 160 μg/assay) were dried, sonicated in 50 μl of 50 mm HEPES (pH 7.2), 100 mm NaCl and centrifuged for 10 min at 12,000 × g at room temperature. The pellets were resuspended in 50 μl of 50 mm HEPES (pH 7.2) and added to 50 μl of TBS containing 0.1 mg/ml purified recombinant GST-C2 domains and EGTA (2 mm final concentration) or CaCl2 (1 mm final concentration). After incubation for 15 min at room temperature, the samples were centrifuged for 10 min at 12,000 × g. The supernatant was precipitated using 10% trichloroacetic acid and the protein pellets washed 3 times with acetone at −20 °C and boiled in SDS-PAGE sample buffer. The pellets were extracted with acetone for 30 min at −20 °C, centrifuged for 10 min at 12,000 × g at room temperature, and boiled in SDS-PAGE sample buffer. Equivalent amounts of the pellet and the supernatant were analyzed by SDS-PAGE and Coomassie Blue staining. The gels were scanned using a flatbed densitometer (Epson) and the data analyzed using the program Aida 1.20 beta. To analyze binding of 45Ca2+ to the recombinant GST-C2 domains, phospholipids and proteins were prepared as above and 10 μCi of 45CaCl2 (Amersham, 2.2 mCi/ml) added to each sample in the presence of 1 mm unlabeled CaCl2. After incubation for 10 min at room temperature, 10 μl of glutathione-Sepharose were added and the samples incubated with constant rotation for 30 min. The beads were then centrifuged for 20 s at 1000 × g and washed twice in 50 mm HEPES (pH 7.2), 100 mm NaCl. The radioactivity bound to the beads was quantified by scintillation counting. PI 3-kinase activity of the immunoprecipitates was assayed by resuspending them in 25 μl of 2 × kinase buffer (40 mm Tris-HCl (pH 7.4), 200 mm NaCl, 2 mm dithiothreitol). Phospholipids (PI, PI(4)P, and PI(4,5)P2, Sigma) stored in CHCl3/MeOH (1:1) (v/v) were dried, sonicated for 15 min in 50 mm Tris-HCl (pH 7.4), and added at 0.2 mg/ml final concentration to the samples. The reactions (50 μl final volume) were started by the addition of 40 μm ATP and 10 μCi of [γ-32P]ATP (3000 Ci/mmol, Amersham) and 3.5 mm of the relevant divalent cations. In order to determine the kinetic parameters of the enzyme for ATP salts, the immunoprecipitates and the phospholipids were incubated in a 40-μl reaction volume, and various concentrations of Mg-ATP, Ca-ATP, or Na2-ATP (all from Sigma) containing [γ-32P]ATP (0.2 μCi/nmol) added in 10 μl. After incubation for 15 min at 37 °C, 100 μl of 1 n HCl and 200 μl of CHCl3/MeOH (1:1) (v/v) were added. The organic phase was collected, re-extracted with 40 μl of MeOH, 1 nHCl (1:1) (v/v) and radioactivity measured by Cerenkov counting. The samples were then dried, resuspended in 30 μl of CHCl3/MeOH (1:1) (v/v) and spotted onto channelled Silica Gel 60 TLC plates (Whatman), which had been pretreated in 1% (w/v) oxalic acid, 1 mm EDTA, 40% MeOH (v/v) and baked for 15 min at 110 °C. The plates were developed in propanol, 2m acetic acid (65:35) (v/v) and the radiolabeled spots quantified using a PhosphorImager (Molecular Dynamics). These analyses were performed as described in Ref. 45Serunian L.A. Auger K.R. Cantley L.C. Methods Enzymol. 1991; 198: 78-87Crossref PubMed Scopus (130) Google Scholar. Radioactive spots corresponding to specific phospholipids were scraped from the TLC plates and incubated with methylamine (33% (w/w) in EtOH) for 1 h at 53 °C. The samples were dried in a SpeedVac and the silica gel extracted three times with 0.25 ml of H2O. After centrifugation, the supernatant was extracted twice with an equal volume of butanol/petroleum ether/ethyl formate (20:4:1, v/v/v). The aqueous phase containing the deacylated phospholipids was then analyzed on a Partisphere SAX HPLC column (Whatman). A gradient of 0 to 25% B over 60 min followed by 25 to 70% B over 30 min (A: H2O, B: 1 m (NH4)2HPO4 pH 3.8) was used. Radioactive peaks were monitored using a continuous flow scintillation counter (Reeve Analytical, Glasgow, United Kingdom). The retention time of the samples was compared with that of a glycero-PI(3)P standard produced in an in vitro PI kinase assay using purified recombinant bovine p110α and of a glycero-PI(4)P standard produced by an in vitro PI kinase assay using purified membrane preparations of A431 cells in the presence of 0.5% (w/v) Nonidet P-40. The alignment of the C2 domain of PI 3-kinase C2β with other C2 domains was initially obtained using Multalign (46Barton G.J. Sternberg M.J.E. J. Mol. Biol. 1
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