Cross-talk between Insulin Receptor and Integrin α5β1 Signaling Pathways

The ligation and clustering of cell surface αβ heterodimeric integrins enhances cell adhesion and initiates signaling pathways that regulate such processes as cell spreading, migration, differentiation, proliferation and apoptosis. Here we show that insulin treatment of Chinese hamster ovary cells expressing insulin receptors (CHO-T) markedly promotes cell adhesion onto a fibronectin matrix, but not onto bovine serum albumin or poly-lysine. Incubation of cells with a GRGDSP peptide that specifically binds integrins (but not the nonspecific GRADSP peptide) abolishes this insulin effect, as does the potent phosphoinositide 3-kinase (PI 3-kinase) inhibitor wortmannin. Moreover, a specific blocking monoclonal anti-α5β1 integrin antibody, PB-1, blocks insulin-stimulated cell adhesion onto fibronectin. Conversely, activating α5β1 integrins on CHO-T cells by adherence onto fibronectin markedly potentiates the action of insulin to enhance insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation. Activation of α5β1 integrin also markedly potentiates the recruitment of p85-associated PI 3-kinase activity to IRS-1 in response to submaximal levels of insulin in CHO-T cells. These data indicate that insulin potently activates integrin α5β1 mediated CHO-T cell adhesion, while integrin α5β1 signaling in turn enhances insulin receptor kinase activity and formation of complexes containing IRS-1 and PI 3-kinase. These findings raise the hypothesis that insulin receptor and α5β1 integrin signaling act synergistically to enhance cell adhesion. The ligation and clustering of cell surface αβ heterodimeric integrins enhances cell adhesion and initiates signaling pathways that regulate such processes as cell spreading, migration, differentiation, proliferation and apoptosis. Here we show that insulin treatment of Chinese hamster ovary cells expressing insulin receptors (CHO-T) markedly promotes cell adhesion onto a fibronectin matrix, but not onto bovine serum albumin or poly-lysine. Incubation of cells with a GRGDSP peptide that specifically binds integrins (but not the nonspecific GRADSP peptide) abolishes this insulin effect, as does the potent phosphoinositide 3-kinase (PI 3-kinase) inhibitor wortmannin. Moreover, a specific blocking monoclonal anti-α5β1 integrin antibody, PB-1, blocks insulin-stimulated cell adhesion onto fibronectin. Conversely, activating α5β1 integrins on CHO-T cells by adherence onto fibronectin markedly potentiates the action of insulin to enhance insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation. Activation of α5β1 integrin also markedly potentiates the recruitment of p85-associated PI 3-kinase activity to IRS-1 in response to submaximal levels of insulin in CHO-T cells. These data indicate that insulin potently activates integrin α5β1 mediated CHO-T cell adhesion, while integrin α5β1 signaling in turn enhances insulin receptor kinase activity and formation of complexes containing IRS-1 and PI 3-kinase. These findings raise the hypothesis that insulin receptor and α5β1 integrin signaling act synergistically to enhance cell adhesion. insulin receptor substrate phosphoinositide 3-kinase focal adhesion kinase, PI, phosphatidylinositol Chinese hamster ovary bovine serum albumin phosphate-buffered saline polyacrylamide gel electrophoresis thin layer chromatography platelet-derived growth factor protein kinase B. The tyrosine kinase activity of the cell surface insulin receptor is required to mediate its many biological actions. Insulin receptor activation promotes the rapid autophosphorylation of its β subunit, as well as tyrosine phosphorylation of proteins involved in insulin signaling, such as insulin receptor substrate (IRS)1 and Shc proteins (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Sun X.-J. Wang L-M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (764) Google Scholar, 3Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 4Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Insulin-mediated phosphorylation of these proteins is thought to provide tyrosine phosphate docking sites for the recruitment of signaling proteins containing Src homology 2 domains (SH2) (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 5Myers Jr., M.G. Sun X.-J. White M.F. Trends Biochem. Sci. 1994; 19: 289-293Abstract Full Text PDF PubMed Scopus (289) Google Scholar, 6Skolnik 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 (607) Google Scholar). One SH2 domain-containing family of proteins which associates with IRS proteins in response to insulin are isoforms of the p85 regulatory subunit of the p110-type PI 3-kinases (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Sun X.-J. Wang L-M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (764) Google Scholar, 3Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 4Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar, 7Kapeller R. Cantley L. BioEssay. 1994; 16: 565-576Crossref PubMed Scopus (553) Google Scholar). PI 3-kinase activity in such signaling complexes appears to be required for insulin action on many cellular processes, including glucose transport (8Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 9Shepherd P.R. Nave B.T. Siddle K. Biochem. J. 1995; 305: 25-28Crossref PubMed Scopus (232) Google Scholar), glycogen synthesis (9Shepherd P.R. Nave B.T. Siddle K. Biochem. J. 1995; 305: 25-28Crossref PubMed Scopus (232) Google Scholar, 10Cross 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 (420) Google Scholar), stress fiber breakdown (11Martin S.S. Rose D.W. Saltiel A.R. Klippel A. Williams L.T. Olefsk J.M. Endocrinology. 1996; 137: 5045-5054Crossref PubMed Scopus (45) Google Scholar), and membrane ruffling (12Kotani K. Yonezawa K. Hara K. Ueda H. Kitamura Y. Sakaue H. Ando A. Chavanieu A. Calas B. Grigorescu F. Nishiyama M. Waterfield M.D. Kasuga M. EMBO J. 1994; 13: 2313-2321Crossref PubMed Scopus (327) Google Scholar). Thus, inhibition of PI 3-kinase activity by wortmannin or disruption of PI 3-kinase recruitment to IRS proteins by dominant inhibitory constructs of p85 subunits ablate the actions of insulin on these processes (11Martin S.S. Rose D.W. Saltiel A.R. Klippel A. Williams L.T. Olefsk J.M. Endocrinology. 1996; 137: 5045-5054Crossref PubMed Scopus (45) Google Scholar, 12Kotani K. Yonezawa K. Hara K. Ueda H. Kitamura Y. Sakaue H. Ando A. Chavanieu A. Calas B. Grigorescu F. Nishiyama M. Waterfield M.D. Kasuga M. EMBO J. 1994; 13: 2313-2321Crossref PubMed Scopus (327) Google Scholar, 13Quon 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). Our understanding of the downstream elements that mediate the action of the 3′-phosphoinositide products of the PI 3-kinases is incomplete, but appear to include protein kinases such as PDK1 and Akt/protein kinase B (PKB) (14Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1223) Google Scholar), a family of proteins containing Sec7 homology domains (15Klarlund J. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (369) Google Scholar), and the zinc finger containing protein EEA1 (16Patki V. Virbasius J. Lane W.S. Toh B.-H. Shpetner H.S. Corvera S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7326-7330Crossref PubMed Scopus (202) Google Scholar). Insulin action also promotes dephosphorylation of tyrosine phosphates on such proteins as focal adhesion kinase (FAK) and paxillin (17Pillay T.S. Sasaoka T. Olefsky J.M. J. Biol. Chem. 1995; 270: 991-994Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 18Knight J.B. Yamauchi K. Pessin J.E. J. Biol. Chem. 1995; 270: 10199-10203Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 19Ouwens D.M. Mikkers H.M.M. Van Der Zon G.C.M. Stein-Gerlach M. Ullrich A. Maassen J.A. Biochem. J. 1996; 318: 609-614Crossref PubMed Scopus (44) Google Scholar), thought to be involved in cell regulation by integrins. Integrins are αβ heterodimeric transmembrane receptors that mediate interactions between the cell surface and the extracellular matrix and also initiate signaling events (20Dedhar S. Hannigan G.E. Curr. Opin. Cell Biol. 1996; 8: 657-669Crossref PubMed Scopus (344) Google Scholar, 21Clark E.A. Brugge J.S. Science. 1995; 268: 233-238Crossref PubMed Scopus (2812) Google Scholar, 22Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell. Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1467) Google Scholar), including tyrosine phosphorylation of FAK and paxillin (23Richardson A. Parsons J.T. Bioessays. 1995; 17: 229-236Crossref PubMed Scopus (256) Google Scholar, 24Guan J.-L. Int. J. Biochem. Cell Biol. 1997; 29: 1085-1096Crossref PubMed Scopus (278) Google Scholar, 25Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1182) Google Scholar), cytoskeletal reorganization (20Dedhar S. Hannigan G.E. Curr. Opin. Cell Biol. 1996; 8: 657-669Crossref PubMed Scopus (344) Google Scholar, 21Clark E.A. Brugge J.S. Science. 1995; 268: 233-238Crossref PubMed Scopus (2812) Google Scholar, 22Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell. Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1467) Google Scholar, 25Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1182) Google Scholar), activation of mitogen-activated protein kinase cascades (26Chen Q. Kinch M.S. Lin T.H. Burridge K. Juliano R.L. J. Biol. Chem. 1994; 269: 26602-26605Abstract Full Text PDF PubMed Google Scholar), and regulation of gene expression (22Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell. Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1467) Google Scholar, 27Yurochko A.D. Liu D.Y. Eierman D. Haskill S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9034-9038Crossref PubMed Scopus (197) Google Scholar). These biological actions of integrins can overlap with those of growth factors, and there is evidence that signaling pathways initiated by integrins synergize functionally with those triggered by growth factors (22Schwartz M.A. Schaller M.D. Ginsberg M.H. Annu. Rev. Cell. Dev. Biol. 1995; 11: 549-599Crossref PubMed Scopus (1467) Google Scholar, 28Miyamoto S. Teramoto H. Gutkind J.S. Yamada K.M. J. Cell Biol. 1996; 135: 1633-1642Crossref PubMed Scopus (676) Google Scholar). Thus, cell adhesion has been shown to greatly enhance autophosphorylation of epidermal growth factor and platelet-derived growth factor (PDGF) receptors (25Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1182) Google Scholar, 29Cybulsky A.V. McTavish A.J. Cyr M.-D. J. Clin. Invest. 1994; 94: 68-78Crossref PubMed Scopus (58) Google Scholar). This in turn potentiates the action of these growth factors in activating mitogen-activated protein kinases, PI 3-kinase, and the downstream protein kinases PDK1 and Akt/PKB (29Cybulsky A.V. McTavish A.J. Cyr M.-D. J. Clin. Invest. 1994; 94: 68-78Crossref PubMed Scopus (58) Google Scholar, 30King W.G. Mattalano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (385) Google Scholar). The mitogenic effects of insulin have also been shown to be enhanced by interactions of extracellular vitronectin with cell surface αvβ3 integrins, which associate with insulin receptor and IRS-1 in response to insulin (31Vuori K. Ruoslahti E. Science. 1994; 266: 1576-1578Crossref PubMed Scopus (339) Google Scholar, 32Schneller M. Vuori K. Ruoslahti E. EMBO J. 1997; 16: 5600-5607Crossref PubMed Scopus (425) Google Scholar). However, the mechanism of such synergism in the actions of insulin and integrins is still unclear. Conversely, it also has been shown that growth factor receptors, such as KIT (33Serve H. Yee N.S. Stella G. Sepp-Lorenzino L. Tan J.C. Besmer P. EMBO J. 1995; 14: 473-483Crossref PubMed Scopus (199) Google Scholar) and PDGF (34Kinashi T. Escobedo J.A. Williams L.T. Takatsu K. Springer T.A. Blood. 1995; 86: 2086-2090Crossref PubMed Google Scholar) receptors, stimulate integrin-mediated cell adhesion onto fibronectin through a PI 3-kinase-dependent pathway. Thus, in the present studies, we addressed the questions whether insulin may directly activate integrin-mediated cell adhesion and if integrin activation modulates insulin signaling. Here, we show that insulin markedly promotes CHO-T cell adhesion onto a fibronectin matrix by a mechanism that is mediated by α5β1 integrin. Activation of this integrin in turn enhances insulin receptor and IRS-1 tyrosine phosphorylation, as well as recruitment of PI 3-kinase activity to IRS-1 in response to insulin. This cross-talk between insulin and integrin receptor pathways is likely to play an important role in biological processes regulated by insulin and which depend on integrin engagement. Anti-phosphotyrosine (anti-Tyr(p)) mouse monoclonal 4G10, anti-p85 polyclonal, and laminin were purchased from Upstate Biotechnology. Rabbit polyclonal anti-IRS-1 immunoglobulin used for immunoprecipitation was prepared as described previously (35Guilherme A. Klarlund J.K. Krystal G. Czech M.P. J. Biol. Chem. 1996; 271: 29533-29536Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Anti-insulin receptor monoclonal (CT-1) and polyclonal antibodies were from mouse ascites and from Santa Cruz Biotechnology, respectively. Plasma human fibronectin was from Boehringer. GRGDSP and GRADSP peptides for the adhesion competition assay were from Life Technologies, Inc. Specific anti α5β1integrin receptor monoclonal antibody, used in CHO-T cell adhesion blocking assay, was a gift from Dr. Rudolph Juliano. [γ-32P]ATP and 35S-labeled protein labeling mix were from NEN Life Science Products. Phosphatidylinositol (PI) was from Avanti Polar Lipids. CHO-T cells were maintained in Ham's F-12 medium, 10% fetal bovine serum, and 50 μg/ml streptomycin/penicillin and grown to confluence before use. To assay cell adhesion on different matrices, cell culture dishes (12-well plate) were coated with fibronectin (0.5 μg/ml), laminin (10 μg/ml), and poly-lysine (0.5 μg/ml) at 4 °C overnight and blocked with 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 1 h, prior to plating the cells. Confluent CHO-T cells were serum for in F-12 containing and protein labeling mix plate) at °C and with with were by were in for and were for the in the were in dishes coated with different as cells were and cells were with containing cells were by and were by a in or on a were by 2 1 μg/ml μg/ml and on for were by at for in the and protein was by the protein assay from antibodies were to the cell for cell and the were overnight at 4 °C by was and the for 2 proteins were in with by gel electrophoresis and to for with monoclonal or polyclonal antibodies were by the Life Science with at The tyrosine phosphorylation levels of insulin receptor and IRS-1 were by PI 3-kinase activity were in by immunoprecipitation of IRS-1 from cell as described The IRS-1 were in in containing 1 and in PI 3-kinase assay The IRS-1 were in of PI 3-kinase assay containing 50 of PI and [γ-32P]ATP at of and the was for at and with 1 were with with 1 and by thin layer chromatography and the products were as described previously (35Guilherme A. Klarlund J.K. Krystal G. Czech M.P. J. Biol. Chem. 1996; 271: 29533-29536Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). To whether insulin signaling can activate integrin-mediated cell adhesion, CHO-T cells were from culture with insulin for at and onto dishes coated with bovine serum or fibronectin. shown in 1 adhesion of CHO-T cells onto a fibronectin matrix was by prior treatment with insulin. was a laminin matrix was used adhesion processes were not to be by insulin insulin treatment not have on CHO-T cell adhesion to dishes coated with bovine serum albumin or poly-lysine. Thus, insulin can CHO-T cell adhesion, to the effects described for growth factor receptors, such as those that and KIT (33Serve H. Yee N.S. Stella G. Sepp-Lorenzino L. Tan J.C. Besmer P. EMBO J. 1995; 14: 473-483Crossref PubMed Scopus (199) Google Scholar, T. Escobedo J.A. Williams L.T. Takatsu K. Springer T.A. Blood. 1995; 86: 2086-2090Crossref PubMed Google of anti-α5β1 blocking on CHO-T cell adhesion onto fibronectin and 35S-labeled and CHO-T cells were and at 4 °C with μg/ml of anti-α5β1 integrin blocking monoclonal 1 of cells were or not with insulin for at °C and on fibronectin (0.5 μg/ml) or μg/ml) as and to for or respectively. are the of whether the action of insulin to cell adhesion to fibronectin was mediated by integrins. Thus, the of a specific GRGDSP which binds to integrins K.M. J. Biol. Chem. 266: Full Text PDF PubMed Google Scholar), on insulin-stimulated CHO-T cell adhesion to fibronectin was in 1 the of GRGDSP peptide to assay and insulin-stimulated cell adhesion to an GRADSP peptide at the Thus, these data that insulin action modulates integrin-mediated CHO-T cell adhesion. has previously been that cells to fibronectin by α5β1 integrin receptors Juliano R.L. Science. PubMed Scopus Google Scholar). the of a monoclonal anti-α5β1 blocking antibody, Juliano R.L. Science. PubMed Scopus Google Scholar, Juliano R.L. J. Cell Biol. 1993; Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar), on CHO-T cell adhesion to fibronectin in the and of insulin. in 2 of to assay markedly the adhesion of CHO-T cells to a fibronectin matrix and insulin-stimulated inhibition of CHO-T cell adhesion onto laminin be by to the assay These data are with a inhibition of cell adhesion onto but not onto laminin Juliano R.L. Science. PubMed Scopus Google Scholar). the data in 1 and 2 a of insulin to enhance CHO-T cell adhesion onto fibronectin through α5β1 integrin. To whether insulin-stimulated and integrin-mediated cell adhesion is on CHO-T cells were from the culture with or 50 of the PI 3-kinase inhibitor wortmannin for prior to with or insulin for and onto fibronectin or in wortmannin treatment of the cells markedly CHO-T cell adhesion. were cells were onto dishes These that insulin-stimulated cell adhesion in this is at in on PI 3-kinase The mechanism of this insulin action on cell adhesion may elements with the actions of growth factors such as PDGF on adhesion, which are also blocked by wortmannin (33Serve H. Yee N.S. Stella G. Sepp-Lorenzino L. Tan J.C. Besmer P. EMBO J. 1995; 14: 473-483Crossref PubMed Scopus (199) Google Scholar, T. Escobedo J.A. Williams L.T. Takatsu K. Springer T.A. Blood. 1995; 86: 2086-2090Crossref PubMed Google Scholar, Y. S.W. 1996; 17: Full Text PDF PubMed Scopus Google Scholar). of cells to fibronectin in tyrosine phosphorylation of FAK and paxillin proteins that are with focal adhesion complexes (23Richardson A. Parsons J.T. Bioessays. 1995; 17: 229-236Crossref PubMed Scopus (256) Google Scholar, 24Guan J.-L. Int. J. Biochem. Cell Biol. 1997; 29: 1085-1096Crossref PubMed Scopus (278) Google Scholar, 25Burridge K. Turner C.E. Romer L.H. J. Cell Biol. 1992; 119: 893-903Crossref PubMed Scopus (1182) Google Scholar). it has been shown that integrin-mediated cell promotes in tyrosine phosphorylation of epidermal growth factor and PDGF receptors S. Teramoto H. Gutkind J.S. Yamada K.M. J. Cell Biol. 1996; 135: 1633-1642Crossref PubMed Scopus (676) Google Scholar, 29Cybulsky A.V. McTavish A.J. Cyr M.-D. J. Clin. Invest. 1994; 94: 68-78Crossref PubMed Scopus (58) Google Scholar). To whether the α5β1 integrin modulates insulin receptor and IRS-1 tyrosine phosphorylation in response to we in which CHO-T cells were from the culture in for at with or insulin for and in or onto for shown in 4 CHO-T cell to fibronectin markedly tyrosine phosphorylation of the protein that to FAK as well as cell to fibronectin also markedly insulin receptor and IRS-1 tyrosine phosphorylation and in response to insulin by and that insulin treatment of these cells dephosphorylation of with that insulin FAK tyrosine dephosphorylation (17Pillay T.S. Sasaoka T. Olefsky J.M. J. Biol. Chem. 1995; 270: 991-994Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 18Knight J.B. Yamauchi K. Pessin J.E. J. Biol. Chem. 1995; 270: 10199-10203Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 19Ouwens D.M. Mikkers H.M.M. Van Der Zon G.C.M. Stein-Gerlach M. Ullrich A. Maassen J.A. Biochem. J. 1996; 318: 609-614Crossref PubMed Scopus (44) Google Scholar). To whether α5β1 integrin potentiates signaling by insulin at submaximal the of different of insulin on insulin receptor and IRS-1 tyrosine phosphorylation was in cells in or to onto fibronectin CHO-T cell onto a fibronectin matrix markedly the of insulin to enhance tyrosine phosphorylation of the insulin receptor at to this on insulin receptor phosphorylation, IRS-1 tyrosine phosphorylation in response to of insulin was enhanced by CHO-T cell onto fibronectin of cell adhesion on IRS-1 tyrosine phosphorylation and its with PI CHO-T cells were in for at and or not with different insulin for on or in for and cells were and were with anti-IRS-1 for immunoprecipitation and and anti-p85 antibodies or for PI 3-kinase the of IRS-1 from or cells with different of insulin. The anti-p85 from the indicate IRS-1 and p85 in and IRS-1 were for PI 3-kinase activity as described and PI 3-kinase products were by to phosphatidylinositol (PI are and were and a and to PI 3-kinase activity from and respectively. The data are the from The in insulin-stimulated tyrosine phosphorylation of IRS-1 by CHO-T cell adhesion onto fibronectin of signaling proteins such as PI 3-kinase to IRS-1 may be (1White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, 2Sun X.-J. Wang L-M. Zhang Y. Yenush L. Myers Jr., M.G. Glasheen E. Lane W.S. Pierce J.H. White M.F. Nature. 1995; 377: 173-177Crossref PubMed Scopus (764) Google Scholar, 3Lavan B.E. Lane W.S. Lienhard G.E. J. Biol. Chem. 1997; 272: 11439-11443Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 4Lavan B.E. Fantin V.R. Chang E.T. Lane W.S. Keller S.R. Lienhard G.E. J. Biol. Chem. 1997; 272: 21403-21407Abstract Full Text Full Text PDF PubMed Scopus (288) Google R. Cantley L. BioEssay. 1994; 16: 565-576Crossref PubMed Scopus (553) Google Scholar). Thus, we the of α5β1integrin on the of the p85 subunit of PI 3-kinase with in CHO-T cell adhesion onto fibronectin promotes the of p85 with IRS-1 at submaximal and of insulin. were also to the PI 3-kinase activity with IRS-1 treatment of and CHO-T cells with or of insulin. of α5β1 integrin receptor was with a in CHO-T cell PI 3-kinase activity in IRS-1 and PI 3-kinase activity with IRS-1 was also submaximal or of insulin were with cells with cells. These data are with the hypothesis that activation of α5β1 integrin through cell adhesion onto fibronectin enhanced recruitment of PI 3-kinases to in enhanced activity of these The findings the of insulin receptor of IRS-1 by α5β1integrin the of signaling events downstream of One to be regulated by 3-kinase signaling complexes is cell proliferation L.M. Myers M.G. Sun X.-J. White M.F. Pierce J.H. Science. 1993; PubMed Scopus Google Scholar), and evidence that integrin-mediated cell adhesion enhances this insulin action (31Vuori K. Ruoslahti E. Science. 1994; 266: 1576-1578Crossref PubMed Scopus (339) Google Scholar). PI 3-kinase activation appears to also be required for the actions of insulin (8Okada T. Kawano Y. Sakakibara T. Hazeki O. Ui M. J. Biol. Chem. 1994; 269: 3568-3573Abstract Full Text PDF PubMed Google Scholar, 9Shepherd P.R. Nave B.T. Siddle K. Biochem. J. 1995; 305: 25-28Crossref PubMed Scopus (232) Google Scholar, 10Cross 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 (420) Google Scholar, 13Quon 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), it be important in to whether integrin ligation may such insulin The present for the that insulin cell adhesion, and this is PI 3-kinase activity Cell adhesion is also enhanced ligation and clustering of integrins on the cell Thus, findings are with the hypothesis that of the insulin signaling through 3-kinase by integrins an mechanism to enhance adhesion that directly by the integrins. this hypothesis whether 3-kinase complexes in response to integrin activation mediate cell adhesion. also be important in the of insulin regulation of cell adhesion. Dr. Rudolph Juliano for a gift of anti-α5β1 integrin receptor monoclonal blocking and for in the of this