Calmodulin Activates Phosphatidylinositol 3-Kinase

Abstract

Calmodulin and phosphatidylinositol 3-kinase are vital components of a number of common intracellular events. Calmodulin, a ubiquitous Ca2+-dependent effector protein, regulates multiple processes in eukaryotic cells, including cytoskeletal organization, vesicular trafficking, and mitogenesis. Phosphatidylinositol 3-kinase participates in events downstream of the receptors for insulin and other growth factors. Here we demonstrate by coimmunoprecipitation and affinity chromatography that Ca2+/calmodulin associates with Src homology 2 domains in the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, thereby significantly enhancing phosphatidylinositol 3-kinase activity in vitro and in intact cells. Furthermore, CGS9343B, a calmodulin antagonist, inhibited basal and Ca2+-stimulated phosphorylation of phosphatidylinositol in intact cells. These data demonstrate a novel mechanism for modulating phosphatidylinositol 3-kinase and provide a direct link between components of two fundamental signaling pathways. Calmodulin and phosphatidylinositol 3-kinase are vital components of a number of common intracellular events. Calmodulin, a ubiquitous Ca2+-dependent effector protein, regulates multiple processes in eukaryotic cells, including cytoskeletal organization, vesicular trafficking, and mitogenesis. Phosphatidylinositol 3-kinase participates in events downstream of the receptors for insulin and other growth factors. Here we demonstrate by coimmunoprecipitation and affinity chromatography that Ca2+/calmodulin associates with Src homology 2 domains in the 85-kDa regulatory subunit of phosphatidylinositol 3-kinase, thereby significantly enhancing phosphatidylinositol 3-kinase activity in vitro and in intact cells. Furthermore, CGS9343B, a calmodulin antagonist, inhibited basal and Ca2+-stimulated phosphorylation of phosphatidylinositol in intact cells. These data demonstrate a novel mechanism for modulating phosphatidylinositol 3-kinase and provide a direct link between components of two fundamental signaling pathways. A critical aspect of protein and/or lipid kinase regulation is the tight coupling of catalytic activity to various extracellular or intracellular signals. Alterations in levels of intracellular free Ca2+([Ca2+] i ) 1The abbreviations used are: [Ca2+] i, intracellular free Ca2+; CaM, calmodulin; PI3-kinase, phosphatidylinositol 3-kinase; CHO, Chinese hamster ovary; IRS, insulin receptor substrate; 32D/IRS-1, 32D cells expressing rat IRS-1; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; HPLC, high pressure liquid chromatography; PtdIns-P, phosphatidylinositol phosphate; PtdIns-3-P, phosphatidylinositol 3-phosphate; PtdIns-3,4-P2, phosphatidylinositol 3,4-diphosphate; PtdIns-3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns-4-P, phosphatidylinositol 4-phosphate; PtdIns-4,5-P2, phosphatidylinositol 4,5-diphosphate. 1The abbreviations used are: [Ca2+] i, intracellular free Ca2+; CaM, calmodulin; PI3-kinase, phosphatidylinositol 3-kinase; CHO, Chinese hamster ovary; IRS, insulin receptor substrate; 32D/IRS-1, 32D cells expressing rat IRS-1; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; HPLC, high pressure liquid chromatography; PtdIns-P, phosphatidylinositol phosphate; PtdIns-3-P, phosphatidylinositol 3-phosphate; PtdIns-3,4-P2, phosphatidylinositol 3,4-diphosphate; PtdIns-3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns-4-P, phosphatidylinositol 4-phosphate; PtdIns-4,5-P2, phosphatidylinositol 4,5-diphosphate.mediate the action of several hormones through the regulation of key enzymes; these signaling events are often orchestrated by the Ca2+ effector, calmodulin (CaM) (1Cohen P. Klee C.B. Calmodulin. Elsevier Science Publishing Co., Inc., New York1988Google Scholar). Activation of phosphatidylinositol 3-kinase (PI3-kinase) in many tissues and cell types is required for mitogenesis, neuronal differentiation, and enhanced glucose transport (2Kappeler R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar). PI3-kinase and CaM are common components of several fundamental intracellular processes. For example, wortmannin, an inhibitor of PI3-kinase, and CGS9343B impede insulin-induced glucose uptake (3Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 4Yeh J.I. Gulve E.A. Rameh L. Birnbaum M.J. J. Biol. Chem. 1995; 270: 2107-2111Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 5Shashkin P. Koshkin A. Langley D. Ren J.M. Westerblad H. Katz A. J. Biol. Chem. 1995; 270: 25613-25618Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Both CaM (6Apodaca G. Enrich C. Mostov K.E. J. Biol. Chem. 1994; 269: 19005-19013Abstract Full Text PDF PubMed Google Scholar, 7Chapin S.J. Enrich C. Aroeti B. Havel R.J. Mostov K.E. J. Biol. Chem. 1996; 271: 1336-1342Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and PI3-kinase (8Jones A.T. Clague M.J. Biochem. J. 1995; 311: 31-34Crossref PubMed Scopus (108) Google Scholar) participate in early endosome fusion. In the cytoskeleton, PI3-kinase has been linked to actin rearrangement (9Martin S.S. Haruta T. Morris A.J. Klippel A. Williams L.T. Olefsky J.M. J. Biol. Chem. 1996; 271: 17605-17608Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), binds α-, β- and γ-tubulin (10Kapeller R. Toker A. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1995; 270: 25985-25991Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and plays a role in platelet-derived growth factor- and insulin-induced membrane ruffling (11Wennstrom S. Hawkins P. 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 (391) Google Scholar, 12Ridley A.J. BioEssays. 1994; 16: 321-327Crossref PubMed Scopus (154) Google Scholar). Overexpression of CaM alters cell morphology and the arrangement of microfilaments within the cell (13Rasmussen C.D. Means A.R. Cell Motil. Cytoskeleton. 1992; 21: 45-57Crossref PubMed Scopus (12) Google Scholar). In addition, CaM binds to a variety of cytoskeletal proteins (1Cohen P. Klee C.B. Calmodulin. Elsevier Science Publishing Co., Inc., New York1988Google Scholar) including the family of unconventional myosins (14Hasson T. Mooseker M.S. J. Biol. Chem. 1996; 271: 16431-16434Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and has been implicated in osteoclast membrane ruffling (15Radding W. Williams J.P. Hardy R.W. McDonald J.M. Whitaker C.H. Turbat-Herrera E.A. Blair H.C. J. Cell. Physiol. 1994; 160: 17-28Crossref PubMed Scopus (30) Google Scholar) and the formation of microspikes in neuronal cells (16Davidkova G. Zhang S.P. Nichols R.A. Weiss B. Neuroscience. 1996; 75: 1003-1019Crossref PubMed Scopus (28) Google Scholar). Finally, both PI3-kinase (17Kotani K. Carozzi A.J. Sakaue H. Hara K. Robinson L.J. Clark S.F. Yonezawa K. James D.E. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 209: 343-348Crossref PubMed Scopus (144) Google Scholar) and CaM, via modulation of the association of IQGAP1 with Cdc42 (18Joyal J.L. Annan R.S. Ho Y. Huddleston M.E. Carr S.A. Hart M.J. Sacks D.B. J. Biol. Chem. 1997; 272: 15419-15425Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), may participate in the regulation of Rho family GTPases. Since CaM and PI3-kinase modulate similar cellular events, we evaluated a possible interaction between these two signaling components.RESULTS AND DISCUSSIONThe 85-kDa regulatory subunit of PI3-kinase (p85) was precipitated from lysates of baculovirus-infected Sf9 cells expressing p85 with CaM-Sepharose but not with Sepharose alone, demonstrating an interaction between these two proteins (Fig.1 A). To identify the CaM-binding region of p85, CaM-Sepharose was incubated with GST fusion proteins containing various regions of p85. Probing with anti-GST antibody disclosed that the carboxyl-terminal SH2 domain bound to the CaM-Sepharose (Fig. 1 B). Longer exposure of the blot revealed association of the amino-terminal SH2 domain with CaM-Sepharose, suggesting that this region also interacts with CaM albeit with a lower affinity (data not shown). Binding to either SH2 domain was significantly reduced when Ca2+ was chelated with EGTA (data not shown). Furthermore, endogenous p85 from CHO cell lysates bound to CaM-Sepharose only in the presence of Ca2+(Fig. 1 C). No binding of CaM to the SH3 or breakpoint cluster homology regions was detected.Insulin activates PI3-kinase by inducing the association of the SH2 domains of p85 with specific phosphotyrosine-containing motifs of IRS proteins (19Sun X.J. Miralpeix M. Myers Jr., M.G. Glasheen E.M. Backer J.M. Kahn C.R. White M.F. J. Biol. Chem. 1992; 267: 22662-22672Abstract Full Text PDF PubMed Google Scholar). It has been demonstrated that a phosphorylated YMXM peptide (pY608), derived from amino acids 605–615 of IRS-1, activates PI3-kinase by binding to its SH2 domains (23Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. X.J. M. P. B. J. White M.F. J. 1992; PubMed Scopus Google Scholar). with but not with the peptide in of p85 from CaM-Sepharose (Fig. 1 These data that CaM binds in or the by phosphorylated YMXM the that may from this are that CaM may PI3-kinase or may modulate PI3-kinase activity by with proteins for binding to the SH2 domains of this the PI3-kinase activity with CaM was binding of PI3-kinase from CHO cell lysates to CaM-Sepharose was Ca2+-dependent A). and p85 and the catalytic subunit of PI3-kinase, from CHO cell lysates (Fig. 2 p85 was not with an antibody or B). p85 and in in the PI3-kinase activity in the was that in (Fig. 2 C). These with the of catalytic subunit bound to CaM, the that CaM significantly PI3-kinase of the by PI3-kinase in the revealed that was phosphatidylinositol (Fig. 2 of the phosphatidylinositol kinase activity in CHO cell lysates was phosphatidylinositol (data not a specific interaction of CaM and Furthermore, the PI3-kinase inhibitor R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) the PI3-kinase activity in the by the of this of p85 and PI3-kinase activity from CHO cells. CHO cell lysates incubated with PI3-kinase activity was by the with and and the by is with the of of CHO cells and of protein with or antibody and with antibody to p85 or and with a of of the p85 and of PI3-kinase are PI3-kinase activity in the and was is with the of of from the was from the and by of and is of CHO cell lysates with or and PI3-kinase activity was In data from two are CaM binds to in a D.J. J.L. White M.F. Sacks D.B. 1996; PubMed Scopus Google Scholar), the of a between CaM, p85, and was evaluated in 32D cells, endogenous and Myers Jr., M.G. X.J. S.A. White M.F. PubMed Scopus Google Scholar). of p85 from lysates of 32D and cells precipitated with CaM-Sepharose that the binding is of IRS of p85 from 32D and cells. 32D and cells and incubated with Sepharose or CaM-Sepharose in the presence of by and to PVDF, and for p85. data are of two of PI3-kinase by or CaM was of CHO cell In CaM PI3-kinase activity by enhanced PI3-kinase activity by (Fig. EGTA not significantly basal or PI3-kinase activity in the (data not but the of PI3-kinase by CaM (Fig. These data that CaM PI3-kinase in a Ca2+-dependent activity of PI3-kinase was by CaM (data not shown). the of PI3-kinase by CaM was to that by of the SH2 domains with a that the binding of CaM to the SH2 domains of p85 is a novel mechanism for the regulation of PI3-kinase of activity was in with various of CaM or Ca2+ was in chelated with the are the of PI3-kinase activity to activates PI3-kinase in [Ca2+] i was with the D.J. J.L. White M.F. Sacks D.B. 1996; PubMed Scopus Google Scholar) in CHO cells with and by and phosphatidylinositol and by with by CaM antagonist, CGS9343B, basal levels of and the by [Ca2+] i the formation of phosphatidylinositol was not in from and this was by with Phosphatidylinositol the in to insulin and other growth (2Kappeler R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar, A. C. Cantley L.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, R. White M.F. Cantley L.C. S. A. PubMed Scopus Google Scholar), was not of phosphatidylinositol but not phosphatidylinositol was also enhanced by [Ca2+] i and was to CGS9343B activity may by a similar to that in D. C. Biochem. J. 1994; PubMed Scopus Google levels in CHO are are not are not in a a novel direct interaction between and the SH2 domains of p85 in the of [Ca2+] i, through CaM, PI3-kinase activity in intact cells. the specific phosphatidylinositol by from by cells with insulin or growth (2Kappeler R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar, A. C. Cantley L.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, R. White M.F. Cantley L.C. S. A. PubMed Scopus Google Scholar). demonstrate that in [Ca2+] i PI3-kinase in a from the an of of this fundamental the of that are by and the between intracellular signaling pathways. A critical aspect of protein and/or lipid kinase regulation is the tight coupling of catalytic activity to various extracellular or intracellular signals. Alterations in levels of intracellular free Ca2+([Ca2+] i ) 1The abbreviations used are: [Ca2+] i, intracellular free Ca2+; CaM, calmodulin; PI3-kinase, phosphatidylinositol 3-kinase; CHO, Chinese hamster ovary; IRS, insulin receptor substrate; 32D/IRS-1, 32D cells expressing rat IRS-1; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; HPLC, high pressure liquid chromatography; PtdIns-P, phosphatidylinositol phosphate; PtdIns-3-P, phosphatidylinositol 3-phosphate; PtdIns-3,4-P2, phosphatidylinositol 3,4-diphosphate; PtdIns-3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns-4-P, phosphatidylinositol 4-phosphate; PtdIns-4,5-P2, phosphatidylinositol 4,5-diphosphate. 1The abbreviations used are: [Ca2+] i, intracellular free Ca2+; CaM, calmodulin; PI3-kinase, phosphatidylinositol 3-kinase; CHO, Chinese hamster ovary; IRS, insulin receptor substrate; 32D/IRS-1, 32D cells expressing rat IRS-1; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; HPLC, high pressure liquid chromatography; PtdIns-P, phosphatidylinositol phosphate; PtdIns-3-P, phosphatidylinositol 3-phosphate; PtdIns-3,4-P2, phosphatidylinositol 3,4-diphosphate; PtdIns-3,4,5-P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns-4-P, phosphatidylinositol 4-phosphate; PtdIns-4,5-P2, phosphatidylinositol 4,5-diphosphate.mediate the action of several hormones through the regulation of key enzymes; these signaling events are often orchestrated by the Ca2+ effector, calmodulin (CaM) (1Cohen P. Klee C.B. Calmodulin. Elsevier Science Publishing Co., Inc., New York1988Google Scholar). Activation of phosphatidylinositol 3-kinase (PI3-kinase) in many tissues and cell types is required for mitogenesis, neuronal differentiation, and enhanced glucose transport (2Kappeler R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar). PI3-kinase and CaM are common components of several fundamental intracellular processes. For example, wortmannin, an inhibitor of PI3-kinase, and CGS9343B impede insulin-induced glucose uptake (3Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 4Yeh J.I. Gulve E.A. Rameh L. Birnbaum M.J. J. Biol. Chem. 1995; 270: 2107-2111Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, 5Shashkin P. Koshkin A. Langley D. Ren J.M. Westerblad H. Katz A. J. Biol. Chem. 1995; 270: 25613-25618Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Both CaM (6Apodaca G. Enrich C. Mostov K.E. J. Biol. Chem. 1994; 269: 19005-19013Abstract Full Text PDF PubMed Google Scholar, 7Chapin S.J. Enrich C. Aroeti B. Havel R.J. Mostov K.E. J. Biol. Chem. 1996; 271: 1336-1342Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar) and PI3-kinase (8Jones A.T. Clague M.J. Biochem. J. 1995; 311: 31-34Crossref PubMed Scopus (108) Google Scholar) participate in early endosome fusion. In the cytoskeleton, PI3-kinase has been linked to actin rearrangement (9Martin S.S. Haruta T. Morris A.J. Klippel A. Williams L.T. Olefsky J.M. J. Biol. Chem. 1996; 271: 17605-17608Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar), binds α-, β- and γ-tubulin (10Kapeller R. Toker A. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1995; 270: 25985-25991Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), and plays a role in platelet-derived growth factor- and insulin-induced membrane ruffling (11Wennstrom S. Hawkins P. 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 (391) Google Scholar, 12Ridley A.J. BioEssays. 1994; 16: 321-327Crossref PubMed Scopus (154) Google Scholar). Overexpression of CaM alters cell morphology and the arrangement of microfilaments within the cell (13Rasmussen C.D. Means A.R. Cell Motil. Cytoskeleton. 1992; 21: 45-57Crossref PubMed Scopus (12) Google Scholar). In addition, CaM binds to a variety of cytoskeletal proteins (1Cohen P. Klee C.B. Calmodulin. Elsevier Science Publishing Co., Inc., New York1988Google Scholar) including the family of unconventional myosins (14Hasson T. Mooseker M.S. J. Biol. Chem. 1996; 271: 16431-16434Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) and has been implicated in osteoclast membrane ruffling (15Radding W. Williams J.P. Hardy R.W. McDonald J.M. Whitaker C.H. Turbat-Herrera E.A. Blair H.C. J. Cell. Physiol. 1994; 160: 17-28Crossref PubMed Scopus (30) Google Scholar) and the formation of microspikes in neuronal cells (16Davidkova G. Zhang S.P. Nichols R.A. Weiss B. Neuroscience. 1996; 75: 1003-1019Crossref PubMed Scopus (28) Google Scholar). Finally, both PI3-kinase (17Kotani K. Carozzi A.J. Sakaue H. Hara K. Robinson L.J. Clark S.F. Yonezawa K. James D.E. Kasuga M. Biochem. Biophys. Res. Commun. 1995; 209: 343-348Crossref PubMed Scopus (144) Google Scholar) and CaM, via modulation of the association of IQGAP1 with Cdc42 (18Joyal J.L. Annan R.S. Ho Y. Huddleston M.E. Carr S.A. Hart M.J. Sacks D.B. J. Biol. Chem. 1997; 272: 15419-15425Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), may participate in the regulation of Rho family GTPases. Since CaM and PI3-kinase modulate similar cellular events, we evaluated a possible interaction between these two signaling AND DISCUSSIONThe 85-kDa regulatory subunit of PI3-kinase (p85) was precipitated from lysates of baculovirus-infected Sf9 cells expressing p85 with CaM-Sepharose but not with Sepharose alone, demonstrating an interaction between these two proteins (Fig.1 A). To identify the CaM-binding region of p85, CaM-Sepharose was incubated with GST fusion proteins containing various regions of p85. Probing with anti-GST antibody disclosed that the carboxyl-terminal SH2 domain bound to the CaM-Sepharose (Fig. 1 B). Longer exposure of the blot revealed association of the amino-terminal SH2 domain with CaM-Sepharose, suggesting that this region also interacts with CaM albeit with a lower affinity (data not shown). Binding to either SH2 domain was significantly reduced when Ca2+ was chelated with EGTA (data not shown). Furthermore, endogenous p85 from CHO cell lysates bound to CaM-Sepharose only in the presence of Ca2+(Fig. 1 C). No binding of CaM to the SH3 or breakpoint cluster homology regions was detected.Insulin activates PI3-kinase by inducing the association of the SH2 domains of p85 with specific phosphotyrosine-containing motifs of IRS proteins (19Sun X.J. Miralpeix M. Myers Jr., M.G. Glasheen E.M. Backer J.M. Kahn C.R. White M.F. J. Biol. Chem. 1992; 267: 22662-22672Abstract Full Text PDF PubMed Google Scholar). It has been demonstrated that a phosphorylated YMXM peptide (pY608), derived from amino acids 605–615 of IRS-1, activates PI3-kinase by binding to its SH2 domains (23Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. X.J. M. P. B. J. White M.F. J. 1992; PubMed Scopus Google Scholar). with but not with the peptide in of p85 from CaM-Sepharose (Fig. 1 These data that CaM binds in or the by phosphorylated YMXM the that may from this are that CaM may PI3-kinase or may modulate PI3-kinase activity by with proteins for binding to the SH2 domains of this the PI3-kinase activity with CaM was binding of PI3-kinase from CHO cell lysates to CaM-Sepharose was Ca2+-dependent A). and p85 and the catalytic subunit of PI3-kinase, from CHO cell lysates (Fig. 2 p85 was not with an antibody or B). p85 and in in the PI3-kinase activity in the was that in (Fig. 2 C). These with the of catalytic subunit bound to CaM, the that CaM significantly PI3-kinase of the by PI3-kinase in the revealed that was phosphatidylinositol (Fig. 2 of the phosphatidylinositol kinase activity in CHO cell lysates was phosphatidylinositol (data not a specific interaction of CaM and Furthermore, the PI3-kinase inhibitor R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) the PI3-kinase activity in the by the of this CaM binds to in a D.J. J.L. White M.F. Sacks D.B. 1996; PubMed Scopus Google Scholar), the of a between CaM, p85, and was evaluated in 32D cells, endogenous and Myers Jr., M.G. X.J. S.A. White M.F. PubMed Scopus Google Scholar). of p85 from lysates of 32D and cells precipitated with CaM-Sepharose that the binding is of IRS of p85 from 32D and cells. 32D and cells and incubated with Sepharose or CaM-Sepharose in the presence of by and to PVDF, and for p85. data are of two of PI3-kinase by or CaM was of CHO cell In CaM PI3-kinase activity by enhanced PI3-kinase activity by (Fig. EGTA not significantly basal or PI3-kinase activity in the (data not but the of PI3-kinase by CaM (Fig. These data that CaM PI3-kinase in a Ca2+-dependent activity of PI3-kinase was by CaM (data not shown). the of PI3-kinase by CaM was to that by of the SH2 domains with a that the binding of CaM to the SH2 domains of p85 is a novel mechanism for the regulation of PI3-kinase of activity was in with various of CaM or Ca2+ was in chelated with the are the of PI3-kinase activity to activates PI3-kinase in [Ca2+] i was with the D.J. J.L. White M.F. Sacks D.B. 1996; PubMed Scopus Google Scholar) in CHO cells with and by and phosphatidylinositol and by with by CaM antagonist, CGS9343B, basal levels of and the by [Ca2+] i the formation of phosphatidylinositol was not in from and this was by with Phosphatidylinositol the in to insulin and other growth (2Kappeler R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar, A. C. Cantley L.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, R. White M.F. Cantley L.C. S. A. PubMed Scopus Google Scholar), was not of phosphatidylinositol but not phosphatidylinositol was also enhanced by [Ca2+] i and was to CGS9343B activity may by a similar to that in D. C. Biochem. J. 1994; PubMed Scopus Google levels in CHO are are not are not in a a novel direct interaction between and the SH2 domains of p85 in the of [Ca2+] i, through CaM, PI3-kinase activity in intact cells. the specific phosphatidylinositol by from by cells with insulin or growth (2Kappeler R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar, A. C. Cantley L.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, R. White M.F. Cantley L.C. S. A. PubMed Scopus Google Scholar). demonstrate that in [Ca2+] i PI3-kinase in a from the an of of this fundamental the of that are by and the between intracellular signaling pathways. 85-kDa regulatory subunit of PI3-kinase (p85) was precipitated from lysates of baculovirus-infected Sf9 cells expressing p85 with CaM-Sepharose but not with Sepharose alone, demonstrating an interaction between these two proteins (Fig.1 A). To identify the CaM-binding region of p85, CaM-Sepharose was incubated with GST fusion proteins containing various regions of p85. Probing with anti-GST antibody disclosed that the carboxyl-terminal SH2 domain bound to the CaM-Sepharose (Fig. 1 B). Longer exposure of the blot revealed association of the amino-terminal SH2 domain with CaM-Sepharose, suggesting that this region also interacts with CaM albeit with a lower affinity (data not shown). Binding to either SH2 domain was significantly reduced when Ca2+ was chelated with EGTA (data not shown). Furthermore, endogenous p85 from CHO cell lysates bound to CaM-Sepharose only in the presence of Ca2+(Fig. 1 C). No binding of CaM to the SH3 or breakpoint cluster homology regions was activates PI3-kinase by inducing the association of the SH2 domains of p85 with specific phosphotyrosine-containing motifs of IRS proteins (19Sun X.J. Miralpeix M. Myers Jr., M.G. Glasheen E.M. Backer J.M. Kahn C.R. White M.F. J. Biol. Chem. 1992; 267: 22662-22672Abstract Full Text PDF PubMed Google Scholar). It has been demonstrated that a phosphorylated YMXM peptide (pY608), derived from amino acids 605–615 of IRS-1, activates PI3-kinase by binding to its SH2 domains (23Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. X.J. M. P. B. J. White M.F. J. 1992; PubMed Scopus Google Scholar). with but not with the peptide in of p85 from CaM-Sepharose (Fig. 1 These data that CaM binds in or the by phosphorylated YMXM the that may from this are that CaM may PI3-kinase or may modulate PI3-kinase activity by with proteins for binding to the SH2 domains of p85. To this the PI3-kinase activity with CaM was binding of PI3-kinase from CHO cell lysates to CaM-Sepharose was Ca2+-dependent A). and p85 and the catalytic subunit of PI3-kinase, from CHO cell lysates (Fig. 2 p85 was not with an antibody or B). p85 and in in the PI3-kinase activity in the was that in (Fig. 2 C). These with the of catalytic subunit bound to CaM, the that CaM significantly PI3-kinase of the by PI3-kinase in the revealed that was phosphatidylinositol (Fig. 2 of the phosphatidylinositol kinase activity in CHO cell lysates was phosphatidylinositol (data not a specific interaction of CaM and Furthermore, the PI3-kinase inhibitor R. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) the PI3-kinase activity in the by the of this Since CaM binds to in a D.J. J.L. White M.F. Sacks D.B. 1996; PubMed Scopus Google Scholar), the of a between CaM, p85, and was evaluated in 32D cells, endogenous and Myers Jr., M.G. X.J. S.A. White M.F. PubMed Scopus Google Scholar). of p85 from lysates of 32D and cells precipitated with CaM-Sepharose that the binding is of IRS To of PI3-kinase by or CaM was of CHO cell In CaM PI3-kinase activity by enhanced PI3-kinase activity by (Fig. EGTA not significantly basal or PI3-kinase activity in the (data not but the of PI3-kinase by CaM (Fig. These data that CaM PI3-kinase in a Ca2+-dependent activity of PI3-kinase was by CaM (data not shown). the of PI3-kinase by CaM was to that by of the SH2 domains with a that the binding of CaM to the SH2 domains of p85 is a novel mechanism for the regulation of PI3-kinase To activates PI3-kinase in [Ca2+] i was with the D.J. J.L. White M.F. Sacks D.B. 1996; PubMed Scopus Google Scholar) in CHO cells with and by and phosphatidylinositol and by with by CaM antagonist, CGS9343B, basal levels of and the by [Ca2+] i the formation of phosphatidylinositol was not in from and this was by with Phosphatidylinositol the in to insulin and other growth (2Kappeler R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar, A. C. Cantley L.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, R. White M.F. Cantley L.C. S. A. PubMed Scopus Google Scholar), was not of phosphatidylinositol but not phosphatidylinositol was also enhanced by [Ca2+] i and was to CGS9343B activity may by a similar to that in D. C. Biochem. J. 1994; PubMed Scopus Google Scholar). a novel direct interaction between and the SH2 domains of p85 in the of [Ca2+] i, through CaM, PI3-kinase activity in intact cells. the specific phosphatidylinositol by from by cells with insulin or growth (2Kappeler R. Cantley L.C. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar, A. C. Cantley L.C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, R. White M.F. Cantley L.C. S. A. PubMed Scopus Google Scholar). demonstrate that in [Ca2+] i PI3-kinase in a from the an of of this fundamental the of that are by and the between intracellular signaling pathways. L. Cantley for the p85 GST fusion and B. for the of CGS9343B, S. for the and J. for the