Polyphosphoinositides are thought to be mediators of cellular signaling pathways as well as regulators of cytoskeletal elements and membrane trafficking events. It has recently been demonstrated that a class of phosphatidylinositol (PI) 3,4,5-P3 5′-phosphatases contains SH2 domains and proline-rich regions, which are present in many signaling proteins. We report here that insulin stimulation of Chinese hamster ovary cells (CHO-T) expressing human insulin receptors causes an 8-10-fold increase in PI 3,4,5-P3 5′-phosphatase activity in anti-phosphotyrosine immunoprecipitates of the cell lysates. This insulin-sensitive polyphosphoinositide 5′-phosphatase did not catalyze dephosphorylation of PI 4,5-P2. No change in 5′-phosphatase activity was detected in insulin receptor or IRS-1 immune complexes in response to insulin. However, insulin treatment of CHO-T cells markedly increased the PI 3,4,5-P3 5′-phosphatase activity associated with Shc and Grb2. The insulin-regulated polyphosphoinositide 5′-phosphatase was not immunoreactive with antibody raised against the recently cloned SHIP 5′-phosphatase reported to associate with Shc and Grb2 in B lymphocytes. These data demonstrate that insulin causes formation of complexes containing a PI 3,4,5-P3 5′-phosphatase, and Shc or Grb2, or both, suggesting an important role of this enzyme in insulin signaling. Polyphosphoinositides are thought to be mediators of cellular signaling pathways as well as regulators of cytoskeletal elements and membrane trafficking events. It has recently been demonstrated that a class of phosphatidylinositol (PI) 3,4,5-P3 5′-phosphatases contains SH2 domains and proline-rich regions, which are present in many signaling proteins. We report here that insulin stimulation of Chinese hamster ovary cells (CHO-T) expressing human insulin receptors causes an 8-10-fold increase in PI 3,4,5-P3 5′-phosphatase activity in anti-phosphotyrosine immunoprecipitates of the cell lysates. This insulin-sensitive polyphosphoinositide 5′-phosphatase did not catalyze dephosphorylation of PI 4,5-P2. No change in 5′-phosphatase activity was detected in insulin receptor or IRS-1 immune complexes in response to insulin. However, insulin treatment of CHO-T cells markedly increased the PI 3,4,5-P3 5′-phosphatase activity associated with Shc and Grb2. The insulin-regulated polyphosphoinositide 5′-phosphatase was not immunoreactive with antibody raised against the recently cloned SHIP 5′-phosphatase reported to associate with Shc and Grb2 in B lymphocytes. These data demonstrate that insulin causes formation of complexes containing a PI 3,4,5-P3 5′-phosphatase, and Shc or Grb2, or both, suggesting an important role of this enzyme in insulin signaling. INTRODUCTIONThe insulin receptor belongs to a family of structurally related transmembrane growth factor receptors that exhibit ligand-activated protein-tyrosine kinase activity (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Rosen O.M. Herrera R. Olowe Y Petruzzelli L.M. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3237-3240Crossref PubMed Scopus (304) Google Scholar, 3Yu K.-T. Czech M.P. J. Biol. Chem. 1984; 259: 5277-5286Abstract Full Text PDF PubMed Google Scholar). The insulin receptor kinase activity is thought to be essential for cellular responses to insulin (4Rosen O.M. Science. 1987; 237: 1452-1458Crossref PubMed Scopus (503) Google Scholar, 5Chou C.K. Dull T.J. Russell D.S. Gherzi R. Lebwohl D. Ullrich A. Rosen O.M. J. Biol. Chem. 1987; 262: 1842-1847Abstract Full Text PDF PubMed Google Scholar, 6Ebina Y. Araki E. Taira M. Shimada F. Mori M. Craik C.S. Siddle K. Pierce S.B. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 704-708Crossref PubMed Scopus (267) Google Scholar). Activation of insulin receptor kinase promotes the rapid autophosphorylation of insulin receptor β-subunits as well as tyrosine phosphorylation of several cytoplasmic proteins such as IRS-1, 1The abbreviations used are: IRS-1insulin receptor substrate-1IRinsulin receptorPIphosphatidylinositolSHSrc homologySHIPSH2-containing inositol 5′-phosphataseCHOChinese hamster ovary cellsPBSphosphate-buffered salineBCAbicinchoninic acidPAGEpolyacrylamide gel electrophoresisHPLChigh performance liquid chromatography. Shc, and pp60 that appear to be involved in the insulin signaling pathway (3Yu K.-T. Czech M.P. J. Biol. Chem. 1984; 259: 5277-5286Abstract Full Text PDF PubMed Google Scholar, 7Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1274) Google Scholar, 8Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar, 9Lavan B.E. Lienhard G.E. J. Biol. Chem. 1993; 268: 5921-5928Abstract Full Text PDF PubMed Google Scholar). Evidence indicates that a primary function of the insulin receptor kinase is to place tyrosine phosphate docking sites on these proteins for the recruitment of signaling proteins containing Src homology (SH) 2 domains (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 10Myers M.G. Sun X.-J. White M.F. Trends Biochem. Sci. 1994; 19: 289-293Abstract Full Text PDF PubMed Scopus (288) Google Scholar, 11Skolnik E.Y. Lee C.H. Batzer A. Vicentini L.M. Zhou M. Daly R. Myers Jr., M.J. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (604) Google Scholar). Thus, insulin-induced phosphorylation of IRS-1, Shc, and pp60 promotes their association with specific SH2-containing proteins, which in turn can stimulate the catalytic activity of these SH2 proteins (12Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar, 13Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.-J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar, 14Kuhne M.R. Pawson T. Lienhard G.E. Feng G.-S. J. Biol. Chem. 1993; 268: 11479-11481Abstract Full Text PDF PubMed Google Scholar, 15Sugimoto S. Wandless T.J. Shoelson S.E. Neel B.G. Walsh C.T. J. Biol. Chem. 1994; 269: 13614-13622Abstract Full Text PDF PubMed Google Scholar). One such SH2-containing protein is the p85 regulatory subunit of the p110 phosphatidylinositol (PI) 3-kinase which catalyzes phosphorylation of the 3-position on PI (12Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar, 13Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.-J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar, 16Kapeller R. Cantley L. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar).Strong evidence supports a pivotal role for signaling complexes containing IRS-1 and the p85/p110-type PI 3-kinases in mediating insulin action on GLUT4 glucose transporter redistribution to the plasma membrane leading to increased glucose uptake as well as glycogen synthesis. Inhibition of PI 3-kinase activity by wortmannin (17Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 18Shepherd P.R. Nave B.T. Siddle K. Biochem. J. 1995; 305: 25-28Crossref PubMed Scopus (230) Google Scholar, 19Cross D.A.E. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (419) Google Scholar) or LY294002 (20Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn R.C. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (997) Google Scholar), microinjection of a fusion protein consisting of an SH2 domain of the p85 regulatory subunit of PI 3-kinase (21Haruta T. Morris A.J. Rose D.W. Nelson J.G. Mueckler M. Olefsky J.M. J. Biol. Chem. 1995; 270: 27991-27994Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and disruption of PI 3-kinase recruitment to IRS-1 by dominant inhibitory constructs of p85 (22Quon M.J. Chen H. Ing B.L. Liu M.-L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar) ablate the stimulation of glucose transport and glycogen synthesis by insulin. Expression of IRS-1 antisense RNA in isolated fat cells also inhibits insulin-mediated translocation of epitope-tagged GLUT4 glucose transporters to the cell surface (23Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar). Further, insulin causes the localization of IRS-1·PI 3-kinase complexes to intracellular membrane vesicles containing GLUT4 (24Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), while other growth factors that stimulate PI 3-kinase activity but fail to activate glucose transport do not. 2R. A. Heller-Harrison, M. Morin, A. Guilherme, E. Skolnik, and M. P. Czech, submitted for publication. These data are consistent with the hypothesis that one or more 3′-phosphoinositide species generated in intracellular membranes in response to insulin regulate cellular components involved in membrane trafficking of GLUT4.It is established that the insulin-sensitive p85/p110 PI 3-kinase activity can catalyze formation of PI 3-P, PI 3,4-P2, and PI 3,4,5-P3 from PI, PI 4-P, and PI 4,5-P2, respectively (24Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 25Ruderman N.B. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (391) Google Scholar, 26Kelly K.L. Ruderman N.B. J. Biol. Chem. 1993; 268: 4391-4398Abstract Full Text PDF PubMed Google Scholar). However, no information is available about which of these 3′-phosphoinositide species actually participates in the mechanisms of insulin action. Interestingly, interconversion of these species to the action of and 5′-phosphatases J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, Liu L. P. G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, D.A. Chin Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. EMBO J. 1995; 14: PubMed Scopus Google Scholar, P.A. S. K. R. 1996; PubMed Scopus Google Scholar, Y. Y. Y. J. Biochem. 1996; PubMed Scopus Google Scholar). PI 3,4,5-P3 5′-phosphatases that SH2 and proline-rich domains of signaling proteins Liu L. P. G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, D.A. Chin Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, P.A. S. K. R. 1996; PubMed Scopus Google Scholar). demonstrate the association of PI 3,4,5-P3 5′-phosphatase with Shc Liu L. P. G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, D.A. Chin Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, P.A. S. K. R. 1996; PubMed Scopus Google Scholar). a PI 3,4,5-P3 5′-phosphatase which a with p85/p110 PI 3-kinase in has been M. S. EMBO J. 1995; 14: PubMed Scopus Google Scholar). The of of this PI 3-kinase activity and 5′-phosphatase activity is to cellular PI which be important in cellular These to insulin action also polyphosphoinositide 5′-phosphatase We report here a insulin-mediated recruitment of 5′-phosphatase activity specific for PI 3,4,5-P3 to complexes containing Shc and Grb2. This action of insulin is to an important role in one or more important to this to for polyphosphoinositide 5′-phosphatase activity in of CHO-T cells expressing human insulin of 3,4,5-P3 to and in cell was in the of of the However, of in the PI activity with on the PI 3,4,5-P3 5′-phosphatase This is consistent with a report that is by J. Biol. Chem. Full Text PDF PubMed Google Scholar). Thus, in the of of PI 3,4,5-P3 to PI was as the of the or of the these insulin PI 3,4,5-P3 5′-phosphatase, of CHO-T cells with or insulin with and the as The a increase in PI 3,4,5-P3 5′-phosphatase activity in the to insulin action and insulin receptor and IRS-1 cells with insulin these as by However, insulin receptors or IRS-1 with receptor or antibody no PI 3,4,5-P3 5′-phosphatase activity be detected in the immune complexes and This was also the cell from CHO-T cells not insulin-regulated PI 3,4,5-P3 5′-phosphatase activity present in the phosphate immunoprecipitates was on in the not The immunoprecipitates did not insulin-regulated activity PI was used as not liquid of the was to the in the of tyrosine phosphate immune complexes from and CHO-T cells This demonstrated of PI 3,4,5-P3 to PI in this as well as a increase in PI formation by the immune complexes from cells PI 3,4,5-P3 PI 3,4,5-P3 the of the inositol from CHO-T cells with or insulin with and PI 3,4,5-P3 5′-phosphatase activity was as in are the of the containing PI 3,4,5-P3 with no of immune complexes with immune complexes from cells and as of the of polyphosphoinositide 5′-phosphatases that associate with Shc in response to cell Liu L. P. G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, D.A. Chin Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and that associate with the protein Grb2 in response to B cell to such with the insulin-regulated complexes from of CHO-T cells or PI 3,4,5-P3 5′-phosphatase activity that was that from However, the 5′-phosphatase activity in the and immunoprecipitates was about that associated with the antibody This in the that the and used do not Shc and Grb2 from the cell not the data in demonstrate a of insulin to association of Shc and Grb2 with one or more 5′-phosphatases to PI 3,4,5-P3 but not PI association of PI 3,4,5-P3 5′-phosphatase activity with Shc and Grb2. from CHO-T cells with or insulin for cell with or and immunoprecipitates for PI 3,4,5-P3 5′-phosphatase by with indicates immune the of insulin on PI 3,4,5-P3 5′-phosphatase activity in immune to PI in the and a The data are from by in the of insulin to The in and of this insulin-regulated polyphosphoinositide 5′-phosphatase of the recently cloned 5′-phosphatase that to the domain of Grb2 in and with Shc in response to in cells Liu L. P. G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). This enzyme also for The of antibody Liu L. P. G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar) to SHIP was the insulin-regulated 5′-phosphatase detected in the present CHO-T cell with and the and for PI 3,4,5-P3 5′-phosphatase in the formation of PI in this of hamster with PI 3,4,5-P3 5′-phosphatase in the as well as in the the from hamster a and that was not present in of CHO-T cells not These immune complexes PI 3,4,5-P3 5′-phosphatase activity as demonstrated in Thus, these data that the insulin-regulated polyphosphoinositide 5′-phosphatase is not SHIP However, is that the CHO-T cell 5′-phosphatase is an of this enzyme that is not by the of the insulin-regulated PI 3,4,5-P3 5′-phosphatase is an important insulin-regulated PI 3,4,5-P3 5′-phosphatase is not with from CHO-T cell or hamster cell with and the immunoprecipitates for PI 3,4,5-P3 5′-phosphatase activity as indicates no and indicates PI 3,4,5-P3 5′-phosphatase in immunoprecipitates and from immunoprecipitates from CHO-T cell or hamster lysates. to PI in and a is that the polyphosphoinositide 5′-phosphatases Liu L. P. G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, D.A. Chin Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) and reported here phosphorylation of the of the to catalyze of this is by of PI 3-kinases that exhibit regulatory mechanisms R. Cantley L. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar, Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, S. R. B. R. M.J. J. EMBO J. 1995; 14: PubMed Scopus Google Scholar, J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, the phosphorylation of PI by PI 3-kinases for the 5′-phosphatases specific for PI These by PI 3-kinase and PI 3,4,5-P3 5′-phosphatase in the of PI to PI The that insulin and other growth factor receptor tyrosine regulate of these indicates that of the cellular PI generated in response to these signaling pathways from PI Further, these an important role for PI in signaling by these PI be an that is and specific for one or more signaling pathways such as the protein kinase Nature. 1995; PubMed Scopus Google Scholar). Thus, the PI 3,4,5-P3 5′-phosphatase as a for polyphosphoinositide signaling in which PI 3,4,5-P3 a of from by PI It be important to for cellular proteins that of these association of 5′-phosphatase with Shc and Grb2 in response to insulin reported here a role of this in the Shc and Grb2 are components of complexes containing the factor of that catalyzes of G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar, 11Skolnik E.Y. Lee C.H. Batzer A. Vicentini L.M. Zhou M. Daly R. Myers Jr., M.J. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (604) Google Scholar, G.J. L. J. Bos J.L. Mol. Cell. Biol. 1994; 14: PubMed Google Scholar, S.B. Chen D. S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). and the p110 PI 3-kinase been reported A. A.J. Science. 1995; 268: PubMed Scopus Google Scholar), and in cell a of protein kinase in response to insulin or growth factors is by a of PI 3-kinases S.W. J.M. Biochem. J. 1994; 303: PubMed Scopus Google Scholar). on this are to a signaling function of the insulin-sensitive PI 3,4,5-P3 5′-phosphatase a role in is also PI 3,4,5-P3 generated by PI 3-kinases is a of signaling as R. Cantley L. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar), is to be by the action of the Thus, the signaling of PI 3,4,5-P3 be or by insulin-regulated PI 3,4,5-P3 is to the cellular localization of PI 3,4,5-P3 and to from containing This hypothesis to of cellular of PI the data reported here an important function of PI 3,4,5-P3 5′-phosphatase activity in one or more signaling pathways from the insulin INTRODUCTIONThe insulin receptor belongs to a family of structurally related transmembrane growth factor receptors that exhibit ligand-activated protein-tyrosine kinase activity (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Rosen O.M. Herrera R. Olowe Y Petruzzelli L.M. Cobb M.H. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 3237-3240Crossref PubMed Scopus (304) Google Scholar, 3Yu K.-T. Czech M.P. J. Biol. Chem. 1984; 259: 5277-5286Abstract Full Text PDF PubMed Google Scholar). The insulin receptor kinase activity is thought to be essential for cellular responses to insulin (4Rosen O.M. Science. 1987; 237: 1452-1458Crossref PubMed Scopus (503) Google Scholar, 5Chou C.K. Dull T.J. Russell D.S. Gherzi R. Lebwohl D. Ullrich A. Rosen O.M. J. Biol. Chem. 1987; 262: 1842-1847Abstract Full Text PDF PubMed Google Scholar, 6Ebina Y. Araki E. Taira M. Shimada F. Mori M. Craik C.S. Siddle K. Pierce S.B. Roth R.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 704-708Crossref PubMed Scopus (267) Google Scholar). Activation of insulin receptor kinase promotes the rapid autophosphorylation of insulin receptor β-subunits as well as tyrosine phosphorylation of several cytoplasmic proteins such as IRS-1, 1The abbreviations used are: IRS-1insulin receptor substrate-1IRinsulin receptorPIphosphatidylinositolSHSrc homologySHIPSH2-containing inositol 5′-phosphataseCHOChinese hamster ovary cellsPBSphosphate-buffered salineBCAbicinchoninic acidPAGEpolyacrylamide gel electrophoresisHPLChigh performance liquid chromatography. Shc, and pp60 that appear to be involved in the insulin signaling pathway (3Yu K.-T. Czech M.P. J. Biol. Chem. 1984; 259: 5277-5286Abstract Full Text PDF PubMed Google Scholar, 7Sun X.J. Rothenberg P. Kahn C.R. Backer J.M. Araki E. Wilden P.A. Cahill D.A. Goldstein B.J. White M.F. Nature. 1991; 352: 73-77Crossref PubMed Scopus (1274) Google Scholar, 8Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar, 9Lavan B.E. Lienhard G.E. J. Biol. Chem. 1993; 268: 5921-5928Abstract Full Text PDF PubMed Google Scholar). Evidence indicates that a primary function of the insulin receptor kinase is to place tyrosine phosphate docking sites on these proteins for the recruitment of signaling proteins containing Src homology (SH) 2 domains (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 10Myers M.G. Sun X.-J. White M.F. Trends Biochem. Sci. 1994; 19: 289-293Abstract Full Text PDF PubMed Scopus (288) Google Scholar, 11Skolnik E.Y. Lee C.H. Batzer A. Vicentini L.M. Zhou M. Daly R. Myers Jr., M.J. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (604) Google Scholar). Thus, insulin-induced phosphorylation of IRS-1, Shc, and pp60 promotes their association with specific SH2-containing proteins, which in turn can stimulate the catalytic activity of these SH2 proteins (12Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar, 13Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.-J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar, 14Kuhne M.R. Pawson T. Lienhard G.E. Feng G.-S. J. Biol. Chem. 1993; 268: 11479-11481Abstract Full Text PDF PubMed Google Scholar, 15Sugimoto S. Wandless T.J. Shoelson S.E. Neel B.G. Walsh C.T. J. Biol. Chem. 1994; 269: 13614-13622Abstract Full Text PDF PubMed Google Scholar). One such SH2-containing protein is the p85 regulatory subunit of the p110 phosphatidylinositol (PI) 3-kinase which catalyzes phosphorylation of the 3-position on PI (12Myers Jr., M.G. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10350-10354Crossref PubMed Scopus (381) Google Scholar, 13Backer J.M. Myers Jr., M.G. Shoelson S.E. Chin D.J. Sun X.-J. Miralpeix M. Hu P. Margolis B. Skolnik E.Y. Schlessinger J. White M.F. EMBO J. 1992; 11: 3469-3479Crossref PubMed Scopus (812) Google Scholar, 16Kapeller R. Cantley L. BioEssays. 1994; 16: 565-576Crossref PubMed Scopus (552) Google Scholar).Strong evidence supports a pivotal role for signaling complexes containing IRS-1 and the p85/p110-type PI 3-kinases in mediating insulin action on GLUT4 glucose transporter redistribution to the plasma membrane leading to increased glucose uptake as well as glycogen synthesis. Inhibition of PI 3-kinase activity by wortmannin (17Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 18Shepherd P.R. Nave B.T. Siddle K. Biochem. J. 1995; 305: 25-28Crossref PubMed Scopus (230) Google Scholar, 19Cross D.A.E. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (419) Google Scholar) or LY294002 (20Cheatham B. Vlahos C.J. Cheatham L. Wang L. Blenis J. Kahn R.C. Mol. Cell. Biol. 1994; 14: 4902-4911Crossref PubMed Scopus (997) Google Scholar), microinjection of a fusion protein consisting of an SH2 domain of the p85 regulatory subunit of PI 3-kinase (21Haruta T. Morris A.J. Rose D.W. Nelson J.G. Mueckler M. Olefsky J.M. J. Biol. Chem. 1995; 270: 27991-27994Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), and disruption of PI 3-kinase recruitment to IRS-1 by dominant inhibitory constructs of p85 (22Quon M.J. Chen H. Ing B.L. Liu M.-L. Zarnowski M.J. Yonezawa K. Kasuga M. Cushman S.W. Taylor S.I. Mol. Cell. Biol. 1995; 15: 5403-5411Crossref PubMed Scopus (143) Google Scholar) ablate the stimulation of glucose transport and glycogen synthesis by insulin. Expression of IRS-1 antisense RNA in isolated fat cells also inhibits insulin-mediated translocation of epitope-tagged GLUT4 glucose transporters to the cell surface (23Quon M.J. Butte A.J. Zarnowski M.J. Sesti G. Cushman S.W. Taylor S.I. J. Biol. Chem. 1994; 269: 27920-27924Abstract Full Text PDF PubMed Google Scholar). Further, insulin causes the localization of IRS-1·PI 3-kinase complexes to intracellular membrane vesicles containing GLUT4 (24Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), while other growth factors that stimulate PI 3-kinase activity but fail to activate glucose transport do not. 2R. A. Heller-Harrison, M. Morin, A. Guilherme, E. Skolnik, and M. P. Czech, submitted for publication. These data are consistent with the hypothesis that one or more 3′-phosphoinositide species generated in intracellular membranes in response to insulin regulate cellular components involved in membrane trafficking of GLUT4.It is established that the insulin-sensitive p85/p110 PI 3-kinase activity can catalyze formation of PI 3-P, PI 3,4-P2, and PI 3,4,5-P3 from PI, PI 4-P, and PI 4,5-P2, respectively (24Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 25Ruderman N.B. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (391) Google Scholar, 26Kelly K.L. Ruderman N.B. J. Biol. Chem. 1993; 268: 4391-4398Abstract Full Text PDF PubMed Google Scholar). However, no information is available about which of these 3′-phosphoinositide species actually participates in the mechanisms of insulin action. Interestingly, interconversion of these species to the action of and 5′-phosphatases J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar, R. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, Liu L. P. G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, D.A. Chin Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. EMBO J. 1995; 14: PubMed Scopus Google Scholar, P.A. S. K. R. 1996; PubMed Scopus Google Scholar, Y. Y. Y. J. Biochem. 1996; PubMed Scopus Google Scholar). PI 3,4,5-P3 5′-phosphatases that SH2 and proline-rich domains of signaling proteins Liu L. P. G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, D.A. Chin Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, P.A. S. K. R. 1996; PubMed Scopus Google Scholar). demonstrate the association of PI 3,4,5-P3 5′-phosphatase with Shc Liu L. P. G. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, D.A. Chin Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, P.A. S. K. R. 1996; PubMed Scopus Google Scholar). a PI 3,4,5-P3 5′-phosphatase which a with p85/p110 PI 3-kinase in has been M. S. EMBO J. 1995; 14: PubMed Scopus Google Scholar). The of of this PI 3-kinase activity and 5′-phosphatase activity is to cellular PI which be important in cellular These to insulin action also polyphosphoinositide 5′-phosphatase We report here a insulin-mediated recruitment of 5′-phosphatase activity specific for PI 3,4,5-P3 to complexes containing Shc and Grb2. This action of insulin is to an important role in one or more important to this
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