The Osmotic Shock-induced Glucose Transport Pathway in 3T3-L1 Adipocytes Is Mediated by Gab-1 and Requires Gab-1-associated Phosphatidylinositol 3-Kinase Activity for Full Activation

Abstract

Osmotic shock treatment of 3T3-L1 adipocytes causes an increase in glucose transport activity and translocation of GLUT4 protein similar to that elicited by insulin treatment. Insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, however, activation by osmotic shock was only partially blocked. Additionally, we have found that the newly identified insulin receptor substrate Gab-1 (Grb2-associated binder-1) is tyrosine-phosphorylated following sorbitol stimulation. Treatment of cells with the tyrosine kinase inhibitor genistein inhibited osmotic shock-stimulated Gab-1 phosphorylation as well as shock-induced glucose transport. Furthermore, pretreatment with the selective Src family kinase inhibitor PP2 completely inhibited the ability of sorbitol treatment to cause tyrosine phosphorylation of Gab-1. We have also shown that microinjection of anti-Gab-1 antibody inhibits osmotic shock-induced GLUT4 translocation. Furthermore, phosphorylated Gab-1 binds and activates phosphatidylinositol 3-kinase (PI3K) in response to osmotic shock. The PI3K activity associated with Gab-1 was 82% of that associated with anti-phosphotyrosine antibodies, indicating that Gab-1 is the major site for PI3K recruitment following osmotic shock stimulation. Although wortmannin only causes a partial block of osmotic shock-stimulated glucose uptake, wortmannin completely abolishes Gab-1 associated PI3K activity. This suggests that other tyrosine kinase-dependent pathways, in addition to the Gab-1-PI3K pathway, contribute to osmotic shock-mediated glucose transport. To date, Gab-1 is the first protein identified as a member of the osmotic shock signal transduction pathway. Osmotic shock treatment of 3T3-L1 adipocytes causes an increase in glucose transport activity and translocation of GLUT4 protein similar to that elicited by insulin treatment. Insulin stimulation of GLUT4 translocation and glucose transport activity was completely inhibited by wortmannin, however, activation by osmotic shock was only partially blocked. Additionally, we have found that the newly identified insulin receptor substrate Gab-1 (Grb2-associated binder-1) is tyrosine-phosphorylated following sorbitol stimulation. Treatment of cells with the tyrosine kinase inhibitor genistein inhibited osmotic shock-stimulated Gab-1 phosphorylation as well as shock-induced glucose transport. Furthermore, pretreatment with the selective Src family kinase inhibitor PP2 completely inhibited the ability of sorbitol treatment to cause tyrosine phosphorylation of Gab-1. We have also shown that microinjection of anti-Gab-1 antibody inhibits osmotic shock-induced GLUT4 translocation. Furthermore, phosphorylated Gab-1 binds and activates phosphatidylinositol 3-kinase (PI3K) in response to osmotic shock. The PI3K activity associated with Gab-1 was 82% of that associated with anti-phosphotyrosine antibodies, indicating that Gab-1 is the major site for PI3K recruitment following osmotic shock stimulation. Although wortmannin only causes a partial block of osmotic shock-stimulated glucose uptake, wortmannin completely abolishes Gab-1 associated PI3K activity. This suggests that other tyrosine kinase-dependent pathways, in addition to the Gab-1-PI3K pathway, contribute to osmotic shock-mediated glucose transport. To date, Gab-1 is the first protein identified as a member of the osmotic shock signal transduction pathway. insulin-responsive glucose transporter isoform insulin receptor substrate Grb2-associated binder-1 phosphatidylinositol 3-kinase Dulbecco's modified Eagle's medium phosphate-buffered saline Src homology 2, GTPγS, guanosine 5′-O-(3-thiotriphosphate) tetramethyl-rodamine isothiocyanate polyacrylamide gel electrophoresis Insulin regulates plasma glucose levels primarily through stimulation of glucose uptake into target tissues and suppression of hepatic glucose production. Glucose transport into adipose and skeletal muscle is predominantly induced by translocation of the insulin responsive glucose transporter isoform GLUT41 from an intracellular pool to the plasma membrane (1Holman G.D. Cushman S.W. Bioessays. 1994; 16: 753-759Crossref PubMed Scopus (134) Google Scholar, 2James D.E. Piper R.C. J. Biol. Chem. 1994; 126: 1123-1126Google Scholar, 3Kandror K.V. Pilch P.F. Am. J. Physiol. 1996; 271: E1-E14Crossref PubMed Google Scholar, 4Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar). Although the precise molecular mechanisms and signaling cascades regulating this event have not yet been completely elucidated, recent studies have identified several of the proximal insulin-dependent signaling events. Upon ligand stimulation, the activated insulin receptor tyrosine kinase phosphorylates a variety of intracellular substrates, which are necessary to sort and transmit metabolic or mitogenic signals (5Cheatham B. Kahn C.R. Endoc. Rev. 1995; 16: 117-142Crossref PubMed Google Scholar, 6White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). Recently, Wong and coworkers (7Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (600) Google Scholar) cloned a new member of the IRS protein family, called Gab-1 (Grb2-associated binder-1). Gab-1 is phosphorylated on tyrosine after stimulation with insulin and several growth factors. It possesses 16 potential phosphotyrosine sites, some of which could serve as binding sites for SH2 domains of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), Grb2, phospholipase C-γ, Nck, and SHP-2. This suggests that Gab-1 serves as a docking protein, like the other IRS proteins. However, in contrast to the IRS proteins, Gab-1 does not possess a phosphotyrosine binding domain, which is believed to be involved in direct binding to the insulin receptor (8Rocchi S. Deckert S.T. Murdaca J. Holgado-Madruga M. Wong A.J. Obberghen E. Mol. Endocrinol. 1998; 12: 914-923Crossref PubMed Scopus (65) Google Scholar). Little is known about the physiological roles of Gab-1; however, some evidence suggests that it is a part of the signaling pathways leading to cell growth, transformation, and apoptosis (9Holgado-Madruga M. Moscatello D.K. Emlet D.R. Dieterich R. Wong A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12419-12424Crossref PubMed Scopus (228) Google Scholar). Several studies examining the insulin-stimulated GLUT4 translocation signaling pathway regulating the insulin stimulation of glucose uptake and GLUT4 translocation have demonstrated a role for the activation and/or appropriate targeting of the type I phosphatidylinositol 3-kinase (10Shepherd P.R. Siddle K. Nave B.T. Biochem. Soc. Trans. 1997; 45: 978-981Crossref Scopus (16) Google Scholar, 11Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 25: 471-490Crossref Scopus (837) Google Scholar). Various other stimuli display insulinomimetic properties such as GTPγS or osmotic shock, and can induce translocation of the GLUT4-containing vesicles to the plasma membrane (12Elmendorf J.S. Chen D. Pessin J.E. J. Biol. Chem. 1998; 273: 13289-13296Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 13Haruta T.A. Morris A.J. Vollenweider P. Nelson J.G. Rose D.W. Mueckler M. Olefsky J.M. Endocrinology. 1998; 139: 358-364Crossref PubMed Scopus (22) Google Scholar, 14Wojtaszewski J.F. Laustsen J.L. Derave W. Richter E.A. Biochim. Biophys. Acta. 1998; 1380: 396-404Crossref PubMed Scopus (44) Google Scholar, 15Yeh J.I. Gulve E.A. Rameh L. Birnbaum M.J. J. Biol. Chem. 1995; 270: 2107-2111Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar), and Chen et al. (16Chen D.J. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) have shown that osmotic shock stimulates glucose transport activity and translocation of GLUT4 through a novel tyrosine kinase pathway. In the present work we provide evidence that osmotic shock markedly increases Gab1 tyrosine phosphorylation and Gab-1-associated PI3K activity and that these events are necessary for maximal stimulation of glucose transport. Dulbecco's modified Eagle's medium (DMEM) and Glutamax were obtained from Life Technologies, Inc. Penicillin-streptomycin and fetal calf serum were obtained from Omega Scientific (Tarzana, CA). Insulin was a gift from Eli Lilly (Indianapolis, IN). Silica-coated thin layer chromatography plates and all chemicals, unless otherwise noted, were obtained from Sigma. Gab-1, p85α-N-SH3 antibodies, wortmannin, genistein, and recombinant protein A-agarose were purchased from Upstate Biotechnology (Lake Placid, NY). The GLUT4 antibody was obtained from Chemicon (Temecula, CA). Mouse monoclonal anti-phosphotyrosine (PY20) was from Transduction Laboratories. Sheep immunoglobulin G (IgG) and fluorescein isothiocyanate-, tetramethyl-rodamine isothiocyanate (TRITC)-, and aminomethylcoumarin acetate-conjugated anti-mouse, anti-rabbit, and anti-sheep IgG antibodies were from Jackson Immunoresearch Laboratories Inc. (West Grove, PA). Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz (Santa Cruz, CA). All radioisotopes were obtained from NEN Life Science Products (Boston, MA). Enhanced chemiluminescence reagent was obtained from Pierce. 3T3-L1 fibroblasts were maintained in Dulbecco's modified Eagle's medium, high glucose, containing 10% calf serum. Postconfluency fibroblasts were differentiated into adipocytes by changing the medium with DMEM, high glucose, containing 10% fetal calf serum, 1 μg/ml insulin, 0.1 μg/ml dexamethasone, and 112 μg/ml isobutylmethylxanthine. The medium was removed after 2 days and replaced with DMEM containing 10% fetal calf serum, 5 mm glucose, Glutamax, and 1% penicillin-streptomycin. Seven days after the addition of the differentiation mix, the cells were plated in 6- or 12-well dishes at densities of 8 × 105 and 4 × 105, respectively. The medium was changed every second day until use, 10–12 days postdifferentiation. Approximately 90% of the cells exhibited large lipid droplets indicative of adipocytes. 24 h prior to the start of all experiments, cells were given fresh DMEM containing 10% fetal calf serum, 5 mm glucose without antibiotics. This study protocol was used in all our experiments. Before the uptake 3T3-L1 adipocytes, 12 days postdifferentiation, were placed in DMEM containing 5 mm glucose for 1 h at 37 °C. Cells were then with mm 1 mm 1 mm mm and serum and or as in the Glucose transport was by the addition of 0.1 containing of as A. Li Am. J. Physiol. Google Scholar). uptake was by the addition of 0.1 containing of The was after by and glucose was removed by with phosphate-buffered Cells were in 1 and uptake was by were for protein by protein Cells from were as in the and then with Cells were into mm mm 1 mm with μg/ml 1 μg/ml and Cells were then a was as R.C. James D.E. Am. J. Physiol. PubMed Google Scholar). from were in and by with a 10% The were then to membrane and at 4 with GLUT4 with a secondary peroxidase-conjugated the were by chemiluminescence and plated in dishes were as in the Cells were at 4 in a containing mm mm 1% 4 mm mm mm were at × for at 4 °C. were with Gab-1 antibody and recombinant protein A-agarose at 4 °C. were in was to the and for 5 were by on polyacrylamide were to membrane and with and Gab-1 antibodies to the with peroxidase-conjugated secondary antibodies, were by 3T3-L1 adipocytes were as in the and then at 4 in the were at × for at 4 °C. were with anti-Gab-1 antibody or anti-phosphotyrosine antibody and recombinant protein A-agarose at 4 °C. were by a at × were with 1% and with mm mm and and with mm mm 1 and were in without the R. M.F. Proc. Natl. Acad. Sci. U. S. A. Scholar), PI3K activity was by the phosphorylation of phosphatidylinositol in the of of for The were with of 8 and of and The was removed and to gel thin layer chromatography the of by thin layer chromatography the PubMed Scopus Google Scholar), phosphatidylinositol was by of was used to the 3T3-L1 adipocytes were on day and on in for microinjection on days of GLUT4 was with a microinjection All for microinjection were in microinjection of 5 and mm were into the of the cell with 5 of IgG to of Cells were to for after for with insulin and mm and in in for at the cells were and with fetal calf serum in for The cells were then with antibody in fetal calf serum at 4 °C. the cells were and IgG were by with or IgG by an 3T3-L1 adipocytes were as in the for and then with in for at Cells were then in for 5 in and at for with in with fluorescein antibodies for were in and for and with were by first at cells in Cells that at the were as for membrane The of cells membrane is as the The cells were and with a NY). are as were the of was with studies (16Chen D.J. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), osmotic shock induced by sorbitol treatment in a increase in treatment was not as as insulin, a increase in glucose of that induced by insulin stimulation it is well that activation and/or appropriate intracellular targeting of PI3K is necessary for insulin-stimulated glucose we the role of PI3K in osmotic shock-stimulated pretreatment with the selective PI3K inhibitor wortmannin for completely inhibited insulin-stimulated glucose transport. also in a in osmotic shock-stimulated glucose transport 1 We the for wortmannin of glucose transport 1 insulin-stimulated transport was inhibited by wortmannin in a with a at and an of glucose uptake was also by wortmannin, only to at at To pretreatment with wortmannin GLUT4 translocation to the plasma we plasma from 3T3-L1 adipocytes with insulin or sorbitol in the or of wortmannin by with GLUT4 antibodies 1 Insulin a increase in the translocation of GLUT4 which was completely inhibited by pretreatment with wortmannin Osmotic shock GLUT4 translocation which was inhibited by wortmannin It been that osmotic shock stimulation of 3T3-L1 adipocytes to tyrosine phosphorylation of several in of and with evidence for phosphorylation of the insulin receptor or W. M. S. K. M. M. S. M. Mol. Endocrinol. 1997; PubMed Scopus Google Scholar). Gab-1, a new member of the IRS protein family, in the of on and is tyrosine-phosphorylated after insulin and growth stimulation (7Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (600) Google Scholar, M. Moscatello D.K. Emlet D.R. Dieterich R. Wong A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12419-12424Crossref PubMed Scopus (228) Google Scholar, L. Holgado-Madruga M. D. A. Wong A.J. M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). To Gab-1 is phosphorylated after osmotic shock stimulation, cell from or cells were with anti-Gab-1 antibody and with anti-phosphotyrosine to increase in tyrosine-phosphorylated Gab-1 was sorbitol stimulation with a increase with insulin stimulation insulin sorbitol treatment in the of Gab-1 protein, as by 2, To Gab-1 phosphorylation is tyrosine we first the cells with genistein a tyrosine kinase for by insulin or sorbitol stimulation for with genistein inhibited the and phosphorylation of the Gab-1 protein Furthermore, genistein pretreatment completely insulin and osmotic shock-stimulated glucose transport activity that osmotic shock a tyrosine kinase-dependent pathway to Gab-1 phosphorylation and glucose transport. It is with insulin the insulin receptor is the tyrosine kinase leading to Gab-1 However, for osmotic shock stimulation the is not as Src family can be activated by and to this was a potential of shock induced Gab-1 we the selective Src family kinase inhibitor 3T3-L1 cells were with PP2 by of Gab-1 in this inhibitor completely the ability of sorbitol treatment to cause tyrosine phosphorylation of Gab-1. To the role of Gab-1 protein in osmotic shock-induced glucose we 3T3-L1 adipocytes with an anti-Gab-1 antibody prior to osmotic shock or insulin stimulation and then the translocation of GLUT4 protein to the cell of the anti-Gab-1 antibody markedly inhibited osmotic shock-induced GLUT4 translocation on insulin-stimulated GLUT4 translocation that phosphorylated Gab-1 is necessary for osmotic shock signaling to GLUT4 translocation not for insulin To Gab-1 could serve as a docking protein for PI3K osmotic shock stimulation, we or cell with anti-Gab-1 antibodies and with in sorbitol treatment to a increase in the Gab-1-associated p85 subunit of PI3K with a increase insulin stimulation. were for Gab-1-associated subunit The levels of Gab-1 protein in these was similar not We also Gab-1-associated PI3K activity in osmotic shock-stimulated treatment induced an increase in PI3K activity in anti-Gab-1 with a increase insulin stimulation addition of wortmannin to the completely inhibited Gab-1-associated PI3K activity by osmotic shock or insulin 4 and The of phosphorylated Gab-1 to the activation of PI3K induced by osmotic shock was by the Gab-1-associated PI3K activity to the PI3K activity associated with all proteins. were with Gab-1 antibodies, and the were then with anti-phosphotyrosine The that of the osmotic shock-stimulated PI3K 82% is associated with Gab-1, only of the PI3K activity is associated with Gab-1 after insulin treatment that sorbitol treatment activates PI3K primarily through with tyrosine-phosphorylated Gab-1 protein, this is of for the insulin signaling pathway. membrane is a Morris A.J. A. Olefsky J.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, Rose D.W. A. Olefsky J.M. Endocrinology. 1996; PubMed Scopus Google Scholar, P. A.J. Nelson J.G. M. Rose D.W. Olefsky J.M. Endocrinology. 1997; PubMed Scopus Google Scholar), we PI3K activity by osmotic shock is involved in membrane sorbitol treatment for of the cells cell membrane and In insulin stimulation to the of membrane in of the cells 8 Treatment of cells with wortmannin inhibited by in cells with insulin and by in cells We also the for wortmannin of membrane 8 was inhibited in a with a at at an was with a at at an the several been in the molecular mechanisms of insulin leading to translocation of the GLUT4 glucose transporter from intracellular sites to the plasma membrane in muscle and adipose However, the signaling pathways this Several recent studies have that osmotic shock is an insulinomimetic that increases glucose transport and GLUT4 translocation through a novel tyrosine kinase-dependent pathway molecular as (16Chen D.J. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google W. M. S. K. M. M. S. M. Mol. Endocrinol. 1997; PubMed Scopus Google Scholar). on the of the insulin signaling pathway, we to signal transduction events by insulin and osmotic shock to induce glucose transport and GLUT4 translocation. In our we have identified that are in osmotic shock signal we that Gab-1 protein is an of the osmotic shock-induced glucose transport pathway. we that sorbitol treatment activates PI3K through with tyrosine-phosphorylated Gab-1 protein, which partially glucose transport stimulation. It is that insulin stimulation in the activation of the insulin receptor tyrosine leading to tyrosine phosphorylation of the IRS family of intracellular docking and and activation of PI3K M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar, M.F. Endocrinol. 1995; Full Text PDF PubMed Scopus Google Scholar). Several studies have demonstrated that PI3K and as well as of a of the PI3K regulatory subunit insulin-stimulated GLUT4 translocation and glucose indicating that PI3K activation is necessary for these of insulin Morris A.J. Rose D.W. Nelson J.G. Mueckler M. Olefsky J.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, B. L. L. J. Kahn C.R. Mol. Biol. 1994; PubMed Scopus Google Scholar, M. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). We and have shown that IRS are not necessary for insulin-stimulated glucose transport K. T.A. J.L. Olefsky J.M. Mol. Biol. 1997; PubMed Scopus (65) Google Scholar, A.J. Nelson J.G. Vollenweider P. M. Mueckler M. Rose D.W. Olefsky J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar) and that pathways that the insulin receptor to we have found that can be phosphorylated by the insulin receptor leading to activation of PI3K and stimulation of uptake and GLUT4 translocation Vollenweider P. K. M. K. S. Olefsky J.M. Mol. Biol. PubMed Scopus Google Scholar). Glucose transport in adipocytes and muscle can also be by several other in addition to GLUT4 translocation and glucose uptake in skeletal muscle through an pathway. of such as GTPγS, into 3T3-L1 adipocytes or treatment with glucose uptake and GLUT4 translocation of insulin (12Elmendorf J.S. Chen D. Pessin J.E. J. Biol. Chem. 1998; 273: 13289-13296Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 15Yeh J.I. Gulve E.A. Rameh L. Birnbaum M.J. J. Biol. Chem. 1995; 270: 2107-2111Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar, D.J. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, W. M. S. K. M. M. S. M. Mol. Endocrinol. 1997; PubMed Scopus Google Scholar, J. B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in properties on glucose and activates glucose transport in adipocytes and skeletal muscle (16Chen D.J. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, W. M. S. K. M. M. S. M. Mol. Endocrinol. 1997; PubMed Scopus Google Scholar). insulin, these stimuli not in the activation of the insulin receptor that with the insulin signaling pathway at a glucose uptake, however, been shown to be J.I. Gulve E.A. Rameh L. Birnbaum M.J. J. Biol. Chem. 1995; 270: 2107-2111Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). In the present study we that pretreatment of 3T3-L1 adipocytes with wortmannin partially inhibits osmotic shock-induced glucose the of a pathway as in this The for wortmannin of glucose transport is to the in with and the maximal is only with in respectively. This that and mechanisms a role in osmotic shock-stimulated glucose the of insulin PI3K with the of Chen et al. (16Chen D.J. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), also a for shock stimulation of glucose these were to a The in the for cells that PI3K to wortmannin and/or binding for docking the of glucose transport that we at the of wortmannin be to on in addition to on We have that osmotic shock stimulation of 3T3-L1 adipocytes tyrosine phosphorylation of a new member of the IRS family called Gab-1. It is well that Gab-1 is tyrosine-phosphorylated after insulin or growth stimulation (7Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-564Crossref PubMed Scopus (600) Google M. Moscatello D.K. Emlet D.R. Dieterich R. Wong A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12419-12424Crossref PubMed Scopus (228) Google Scholar, L. Holgado-Madruga M. D. A. Wong A.J. M. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). that tyrosine phosphorylation of Gab-1 induced by osmotic shock is that by insulin, and it is completely by pretreatment with the tyrosine kinase inhibitor It is known that the insulin receptor is the tyrosine kinase that insulin-stimulated Gab-1 tyrosine phosphorylation (8Rocchi S. Deckert S.T. Murdaca J. Holgado-Madruga M. Wong A.J. Obberghen E. Mol. Endocrinol. 1998; 12: 914-923Crossref PubMed Scopus (65) Google Scholar). The precise tyrosine kinase that osmotic shock-stimulated Gab-1 tyrosine phosphorylation is and the of However, Src family can be activated by a variety of we that osmotic shock, in some activates an Src which then serves to Gab-1 in tyrosine with the Src kinase inhibitor PP2 are with this we found that this inhibitor the of sorbitol treatment to cause Gab-1 tyrosine Several of evidence that this osmotic shock-induced phosphorylation of Gab-1 is for stimulation of glucose transport. it is known that the SH2 domains of the p85 subunit of PI3K the M. S. Full Text PDF PubMed Scopus Google Scholar). The growth receptor a which binds to and activates and a in the receptor abolishes growth PI3K activation A. J. PubMed Scopus Google Scholar). Holgado-Madruga et al. (9Holgado-Madruga M. Moscatello D.K. Emlet D.R. Dieterich R. Wong A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12419-12424Crossref PubMed Scopus (228) Google Scholar) that Gab-1 such which could the binding and activation of It also been shown that Gab-1 can as a docking protein for SH2 and can PI3K activation stimulation with growth and insulin (8Rocchi S. Deckert S.T. Murdaca J. Holgado-Madruga M. Wong A.J. Obberghen E. Mol. Endocrinol. 1998; 12: 914-923Crossref PubMed Scopus (65) Google Scholar, M. Moscatello D.K. Emlet D.R. Dieterich R. Wong A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12419-12424Crossref PubMed Scopus (228) Google Scholar). In the we that osmotic shock also stimulates the of Gab-1 with the p85 subunit of PI3K with activation of the In with our Gab-1 tyrosine phosphorylation we also that the of Gab-1-associated PI3K activity by osmotic shock was that by also that the the of the osmotic shock-stimulated PI3K Gab-1-associated PI3K for only a of the insulin-stimulated PI3K activity. Treatment of cells with the tyrosine kinase inhibited shock-stimulated Gab-1 tyrosine as well as stimulation of glucose with the that these events are To this we 3T3-L1 cells with an anti-Gab-1 antibody by osmotic shock treatment and of GLUT4 translocation. We found that of the Gab-1 antibody on insulin-stimulated GLUT4 translocation inhibited osmotic shock-mediated GLUT4 translocation by that Gab-1 is for shock-stimulated glucose transport. We have that activation of PI3K is necessary and to indicating that PI3K the only signaling for insulin to induce Morris A.J. A. Olefsky J.M. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, Rose D.W. A. Olefsky J.M. Endocrinology. 1996; PubMed Scopus Google Scholar, P. A.J. Nelson J.G. M. Rose D.W. Olefsky J.M. Endocrinology. 1997; PubMed Scopus Google Scholar). from our present study the of PI3K activity in osmotic shock-mediated membrane in 3T3-L1 adipocytes. We that sorbitol stimulation is also of membrane only to of the insulin This response is partially by pretreatment with The studies the for Gab-1 in the signaling pathway leading to osmotic shock-stimulated glucose transport and that this pathway PI3K for that PI3K activation in response to osmotic shock, predominantly in with Gab-1, and partially to the stimulation of glucose transport and It is that signaling are to osmotic shock-stimulated glucose uptake and these are only partially by To date, Gab-1 is the first protein identified as a member of the osmotic shock signal transduction pathway. We for the 3T3-L1 adipocytes.