Key points are not available for this paper at this time.
The insulin receptor substrate (IRS) family of proteins mediate a variety of intracellular signaling events by serving as signaling platforms downstream of several receptor tyrosine kinases including the insulin and insulin-like growth factor-1 (IGF-1) receptors. Recently, several new members of this family have been identified including IRS-3, IRS-4, and growth factor receptor-binding protein 2-associated binder-1 (Gab-1). 3T3 cell lines derived from IRS-1-deficient embryos exhibit a 70–80% reduction in IGF-1-stimulated S-phase entry and a parallel decrease in the induction of the immediate-early genes c-fos and egr-1but unaltered activation of the mitogen-activated protein kinases extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2. Reconstitution of IRS-1 expression in IRS-1-deficient fibroblasts by retroviral mediated gene transduction is capable of restoring these defects. Overexpression of Gab-1 in IRS-1-deficient fibroblasts also results in the restoration ofegr-1 induction to levels similar to those achieved by IRS-1 reconstitution and markedly increases IGF-1-stimulated S-phase progression. Gab-1 is capable of regulating these biological end points despite the absence of IGF-1 stimulated tyrosine phosphorylation. These data provide evidence that Gab-1 may serve as a unique signaling intermediate in insulin/IGF-1 signaling for induction of early gene expression and stimulation of mitogenesis without direct tyrosine phosphorylation. The insulin receptor substrate (IRS) family of proteins mediate a variety of intracellular signaling events by serving as signaling platforms downstream of several receptor tyrosine kinases including the insulin and insulin-like growth factor-1 (IGF-1) receptors. Recently, several new members of this family have been identified including IRS-3, IRS-4, and growth factor receptor-binding protein 2-associated binder-1 (Gab-1). 3T3 cell lines derived from IRS-1-deficient embryos exhibit a 70–80% reduction in IGF-1-stimulated S-phase entry and a parallel decrease in the induction of the immediate-early genes c-fos and egr-1but unaltered activation of the mitogen-activated protein kinases extracellular signal-regulated kinase-1 and extracellular signal-regulated kinase-2. Reconstitution of IRS-1 expression in IRS-1-deficient fibroblasts by retroviral mediated gene transduction is capable of restoring these defects. Overexpression of Gab-1 in IRS-1-deficient fibroblasts also results in the restoration ofegr-1 induction to levels similar to those achieved by IRS-1 reconstitution and markedly increases IGF-1-stimulated S-phase progression. Gab-1 is capable of regulating these biological end points despite the absence of IGF-1 stimulated tyrosine phosphorylation. These data provide evidence that Gab-1 may serve as a unique signaling intermediate in insulin/IGF-1 signaling for induction of early gene expression and stimulation of mitogenesis without direct tyrosine phosphorylation. insulin-like growth factor insulin receptor substrate Src homology 2 growth factor receptor-binding protein 2 growth factor receptor-binding protein 2-associated binder-1 pleckstrin homology epidermal growth factor phosphatidylinositol 3-kinase mitogen-activated protein polyacrylamide gel electrophoresis extracellular signal-regulated kinase phosphotyrosine binding Insulin and insulin-like growth factor 1 (IGF-1)1 elicit diverse biological effects by binding to and activating their cognate tyrosine kinase receptors (1.Kasuga M. Karlsson F.A. Kahn C.R. Science. 1982; 215: 185-187Crossref PubMed Scopus (763) Google Scholar, 2.Ullrich A. Gray A. 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Nature. 1995; 377: 173-177Crossref PubMed Scopus (776) Google Scholar). These phosphorylated substrates then bind adapter proteins containing Src homology 2 (SH2) domains, such as the regulatory subunits of PI 3-kinase (6.Backer 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 (832) Google Scholar), protein-tyrosine phosphatase SH2 domain containing phosphatase-2/Syp (7.Sun X.J. Crimmins D.L. Myers Jr., M.G. Miralpeix M. White M.F. Mol. Cell. Biol. 1993; 13: 7418-7428Crossref PubMed Google Scholar), and the growth factor receptor-binding protein 2 (Grb2), which links signaling via SOS to activation of the Ras complex (8.Baltensperger K. Kozma L.M. Cherniack A.D. Klarlund J.K. Chawla A. Banerjee U. Czech M.P. Science. 1993; 260: 1950-1952Crossref PubMed Scopus (233) Google Scholar, 9.Skolnik E.Y. Lee C.H. Batzer A.G. Vicentini L.M. Zhou M. Daly R.J. Myers Jr., M.G. Backer J.M. Ullrich A. White M.F. Schlessinger J. EMBO J. 1993; 12: 1929-1936Crossref PubMed Scopus (610) Google Scholar). In addition, depending on the cell type, the activated insulin and IGF-1 receptors phosphorylate several other substrates, including IRS-3 (10.Hosomi Y. Shii K. Ogawa W. Matsuba H. Yoshida M. Okada Y. Yokono K. Kasuga M. Baba S. Roth R.A. J. Biol. Chem. 1994; 269: 11498-11502Abstract Full Text PDF PubMed Google Scholar, 11.Lavan B.E. Lienhard G.E. J. Biol. Chem. 1993; 268: 5921-5928Abstract Full Text PDF PubMed Google Scholar, 12.Milarski K.L. Lazar D.F. Wiese R.J. Saltiel A.R. Biochem. J. 1995; 308: 579-583Crossref PubMed Scopus (22) Google Scholar, 13.Lavan 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 (293) Google Scholar), IRS-4 (14.Fantin V.R. Sparling J.D. Slot J.W. Keller S.R. Lienhard G.E. Lavan B.E. J. Biol. Chem. 1998; 273: 10726-10732Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), several isoforms of Src homologous and collagen (15.Giorgetti S. Pelicci P.G. Pelicci G. Van Obberghen E. Eur. J. Biochem. 1994; 223: 195-202Crossref PubMed Scopus (100) Google Scholar, 16.Okada S. Yamauchi K. Pessin J.E. J. Biol. Chem. 1995; 270: 20737-20741Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 17.Pelicci G.L. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1198) Google Scholar), and grb-2-associated binder-1 (Gab-1) (18.Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (607) Google Scholar). Together, these intermediate signals stimulate a variety of downstream biological pathways, such as glycogen synthesis, glucose transport, gene expression, and mitogenesis. Gab-1 was initially identified in a cDNA library of glioblastoma tumors based upon its ability to bind Grb-2 (18.Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (607) Google Scholar). Gab-1 shares functional and structural homology with the IRS family of proteins, including an N-terminal pleckstrin homology (PH) domain, several tyrosine phosphorylation sites, and two potential SH3 binding proline-rich regions. Several studies have also demonstrated that Gab-1 functions as a substrate and docking protein downstream in the signaling pathways of several receptor tyrosine kinases, including the receptors for epidermal growth factor (EGF), insulin, fibroblast growth factor, nerve growth factor, hepatocyte growth factor, and various cytokines such as interleukin-3, interleukin-6, interferon-α, and interferon-γ (18.Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (607) Google Scholar, 19.Lehr S. Kotzka J. Herkner A. Klein E. Siethoff C. Knebel B. Noelle V. Bruning J.C. Klein H.W. Meyer H.E. Krone W. Muller-Wieland D. Biochemistry. 1999; 38: 151-159Crossref PubMed Scopus (46) Google Scholar, 20.Yenush L. Makati K.J. Smith-Hall J. Ishibashi O. Myers Jr., M.G. White M.F. J. Biol. Chem. 1996; 271: 24300-24306Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 21.Takahashi-Tezuka M. Yoshida Y. Fukada T. Ohtani T. Yamanaka Y. Nishida K. Nakajima K. Hibi M. Hirano T. Mol. Cell. Biol. 1998; 18: 4109-4117Crossref PubMed Scopus (253) Google Scholar). Like the IRS proteins, tyrosine-phosphorylated Gab-1 has been shown to interact with several Src homologous and collagen-containing adapter molecules, such as the p85 regulatory subunit of PI 3-kinase, SH2 domain containing phosphatase-2, and Grb-2 (18.Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (607) Google Scholar, 21.Takahashi-Tezuka M. Yoshida Y. Fukada T. Ohtani T. Yamanaka Y. Nishida K. Nakajima K. Hibi M. Hirano T. Mol. Cell. Biol. 1998; 18: 4109-4117Crossref PubMed Scopus (253) Google Scholar, 22.Holgado-Madruga M. Moscatello D.K. Emlet D.R. Dietrich R. Wong A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12419-12424Crossref PubMed Scopus (232) Google Scholar). In addition, Gab-1 can interact with phospholipase C-γ, which does not interact with IRS-1. Previous studies in our laboratory have demonstrated a specific role for IRS-1 in insulin and IGF-1-mediated signaling (23.Kahn C.R. Krieger D.T. Bardin C.W. Current Therapy in Endocrinology. B. C. Decker Publishers, Philadelphia1983Google Scholar). Thus, although there is a high degree of homology and structural similarity among the IRS family members, cells with a homozygous deficiency of IRS-1 show a reduced mitogenic response that cannot be recovered by the overexpression of IRS-2 (23.Kahn C.R. Krieger D.T. Bardin C.W. Current Therapy in Endocrinology. B. C. Decker Publishers, Philadelphia1983Google Scholar). These data highlight the potential for specific IRS proteins to uniquely mediate distinct downstream biological events. In the present study, we have investigated the role of Gab-1 in mediating IGF-1 signaling by overexpressing Gab-1 in IRS-1-deficient 3T3 fibroblasts by retroviral mediated gene transfer. Our studies revealed that Gab-1 overexpression produces a unique pattern of downstream signals in IRS-1-deficient fibroblasts, despite an absence of detectable IGF-1-stimulated tyrosine phosphorylation, suggesting a novel role and mechanism for this substrate in insulin/IGF-1 action. Human recombinant IGF-1 was purchased from Peprotech Inc.(Rocky Hill, NJ). The Gab-1 cDNA was a generous gift of Albert Wong (Kimmel Cancer Institute, Philadelphia, PA). Polyclonal antibodies to Gab-1 and IRS-2 and monoclonal antibody to phosphotyrosine were a generous gift from Morris F. White (Joslin Diabetes Center, Boston, MA). Antibodies to MAP kinase and IRS-1 were prepared as B. Wang L. Bruning J.C. 1996; 1 Scholar). The cDNA for was by B. Cancer Institute, Boston, MA). gel electrophoresis were from and were purchased from was from and for and for were purchased from from of were were from the and as as and were The embryos were in the of the embryos was by from the by as E. J.C. Kahn C.R. Nature. 1994; PubMed Scopus Google Scholar). The the was activated for and then by the of of with were then by and a of in with cells were to and then to the 3T3 of and H. J. Biol. PubMed Scopus Google Scholar). The reconstitution of IRS-1 in 3T3 cell lines was achieved as (23.Kahn C.R. Krieger D.T. Bardin C.W. Current Therapy in Endocrinology. B. C. Decker Publishers, Philadelphia1983Google Scholar). Gab-1 in 3T3 cell a retroviral expression was by a and from the of The of the was by and cells were with of were and was to the cells an cells were was by of of Gab-1 was as a was then as for the of stimulation of and cells were in containing and then to IGF-1 a of in with for the were prepared 1 2 2 of for and protein was by in a was by the of The was then in in to with the 1 of protein was with the antibodies for 2 were by of a of protein in for 1 two in containing protein were from by in for were by and to was the were for 2 in by with the antibody the for 2 antibodies were with and of protein was to with a to and and were with protein two in and two in were in of and the were by the of 2 of 2 protein kinase and of protein were to for and by the of of the were for by and to of substrates were with a was to in containing stimulation with IGF-1 for the was by of was to electrophoresis in of the and of the to were with the specific for IRS-1 was by the of to the of IRS-1 specific for IRS-2 was by of the of IRS-2 and the to the of Gab-1 was by of and expression was a cDNA to the for were a an were in is for and for in were and to were a of 2 in 1 the was to with for The cells were then stimulated with the of IGF-1 for and with 2 of for 1 two with cells were for 1 in was then for were in IRS-1-deficient 3T3 cells were from fibroblasts derived from IRS-1-deficient (23.Kahn C.R. Krieger D.T. Bardin C.W. Current Therapy in Endocrinology. B. C. Decker Publishers, Philadelphia1983Google Scholar). these cells have a reduced response to IGF-1-stimulated mitogenesis and early gene expression (23.Kahn C.R. Krieger D.T. Bardin C.W. Current Therapy in Endocrinology. B. C. Decker Publishers, Philadelphia1983Google Scholar). the potential role of Gab-1 in IGF-1-mediated we Gab-1 IRS-1 in these 3T3 cell lines by retroviral mediated gene IRS-1 be in IRS-1 cells a specific for in cells with IRS-1 retroviral mediated gene a of to the IRS-1 be 1 an that cell lines levels of IRS-2 parallel of Gab-1 was in IRS-1 expression a specific for a to Gab-1 was in cells with The of protein expression of and Gab-1 was by on cell of the with an antibody specific to the of IRS-1 the absence of IRS-1 protein in and cells as as the reconstitution of IRS-1 in cell lines cell lines levels of IRS-2 as by with with an antibody the of Gab-1 the of expression of Gab-1 in and cells and a overexpression of Gab-1 with and cell These data the reconstitution of IRS-1 and the overexpression of Gab-1 in and cell and that in the of IRS-2 protein expression from the reconstitution of IRS-1 overexpression of Gab-1 is capable of phosphorylated on tyrosine residues in response to IGF-1 cell from IGF-1 and stimulated and cell lines were to antibody and with In tyrosine phosphorylation of Gab-1 be with rapidly and markedly the of Gab-1 phosphorylation (18.Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (607) Google S. Kotzka J. Herkner A. Klein E. Siethoff C. Knebel B. Noelle V. Bruning J.C. Klein H.W. Meyer H.E. Krone W. Muller-Wieland D. Biochemistry. 1999; 38: 151-159Crossref PubMed Scopus (46) Google Scholar). In addition, this phosphorylation in of the protein on In the of tyrosine phosphorylation was not in response to IGF-1 and of the protein was results were cell prepared from IGF-1-stimulated cell lines were to with antibody by with not with these IGF-1 stimulation to the of Gab-1 with SH2 domain containing adapter proteins such as Grb-2 and the p85 regulatory subunit of PI 3-kinase not the of IGF-1-stimulated IRS-1 and IRS-2 tyrosine phosphorylation and to the absence of tyrosine-phosphorylated Gab-1 in response to cell from IGF-1 stimulated and cells were to and cells IRS-1 tyrosine and the high molecular weight to as by its on In IGF-1 stimulation in a in IRS-1 tyrosine phosphorylation and levels of IRS-2 tyrosine phosphorylation. In IGF-1 stimulation in the tyrosine phosphorylation of IRS-2 to that in tyrosine phosphorylated proteins were in the molecular weight of Gab-1 2 Thus, although Gab-1 is capable of tyrosine phosphorylation in response to stimulation in 3T3 does not tyrosine phosphorylation in response to IGF-1 In addition, these data that the overexpression of Gab-1 does not the ability of IRS-2 to become tyrosine phosphorylated in response to IGF-1 does not to be a substrate for the IGF-1 activated by IGF-1 stimulation and in gene expression and the mitogenic response is the activation of the MAP kinase signaling receptor phosphorylated and bind to the SH2 domain of the adapter molecular which in the factor for activation of Ras and of a signaling of kinases, including mitogen-activated protein kinase and the potential of Gab-1 in mediating signaling via this we in kinase kinase and protein as a can be in the MAP kinases and were activated to the and also the in response to IGF-1 in cell These data that both Gab-1 and although capable of binding Grb-2 and activating the MAP kinases, on the and of IGF-1-stimulated activation of and IRS-1 by gene exhibit and growth of to the of IGF-1 and In IRS-1-deficient 3T3 cells also exhibit a decrease in IGF-1-mediated and c-fos expression (23.Kahn C.R. Krieger D.T. Bardin C.W. Current Therapy in Endocrinology. B. C. Decker Publishers, Philadelphia1983Google Scholar). the potential role for Gab-1 in mediating immediate-early gene expression, IGF-1-stimulated expression of the immediate-early gene was by cells a markedly reduced expression of in response to IGF-1 stimulation a expression be by the reconstitution of IRS-1 in of revealed a reduction ofegr-1 induction in cells with Gab-1 overexpression was also capable of restoring the in expression in cells to levels that of These data the potential of a mediated by both IRS-1 and which is for IGF-1-stimulated activation of the immediate-early (23.Kahn C.R. Krieger D.T. Bardin C.W. Current Therapy in Endocrinology. B. C. Decker Publishers, Philadelphia1983Google Scholar). to overexpression of Gab-1 was capable of restoring IGF-1-stimulated cell we the of IGF-1-stimulated of in and cell in response to IGF-1 was by in and reconstitution of IRS-1 the potential to overexpression of Gab-1 markedly IGF-1-stimulated S-phase in the the of IGF-1 cells a of as with cells and by with results from 3T3 fibroblast cell lines not Thus, Gab-1 is uniquely capable of cell progression. variety of studies have demonstrated that Gab-1 downstream of several receptor protein-tyrosine kinases, including the insulin and by binding SH2 signaling phosphorylation. These studies that Gab-1 functions as a other members of the IRS family of the potential role of Gab-1 other IRS proteins in mediating the effects of insulin and IGF-1 has not been In the study, we have to the potential role of Gab-1 in IGF-1-mediated of cells in which this is reduced by the absence of IRS-1 (23.Kahn C.R. Krieger D.T. Bardin C.W. Current Therapy in Endocrinology. B. C. Decker Publishers, Philadelphia1983Google Scholar). The tyrosine phosphorylation of IRS proteins a mechanism for the with activated insulin and IGF-1 receptors. of IRS-1 the has the N-terminal and of IRS-1 in mediating receptor and this has been in studies in which these have been L. Makati K.J. Smith-Hall J. Ishibashi O. Myers Jr., M.G. White M.F. J. Biol. Chem. 1996; 271: 24300-24306Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, Jr., M.G. J. Glasheen E.M. Wang L.M. Sun X.J. J. Pierce J.H. White M.F. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). on studies in Myers Jr., M.G. J. Glasheen E.M. Wang L.M. Sun X.J. J. Pierce J.H. White M.F. J. Biol. 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Current Therapy in Endocrinology. B. C. Decker Publishers, Philadelphia1983Google Scholar). In addition, the restoration of expression in Gab-1 overexpressing cells that Gab-1 is capable of regulating immediate-early gene These data are of that the IGF-1-stimulated activation of the MAP kinases is in IRS-1-deficient and Our data provide evidence for an activating the despite the activation of the MAP kinases and the cell lines provide a unique with which to the pathways for Gab-1 also to be uniquely capable of IGF-1-stimulated mitogenesis. These results are by data that that the overexpression of Gab-1 is capable of and growth (18.Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (607) Google Scholar). In addition, M. Moscatello D.K. Emlet D.R. Dietrich R. Wong A.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12419-12424Crossref PubMed Scopus (232) Google have shown that cells overexpressing Gab-1 a reduced for the of nerve growth factor to of Gab-1 PI 3-kinase binding levels of that PI 3-kinase was for the of In addition, the overexpression of Gab-1 in several cell to potential that Gab-1 may have a on of the cell these data that Gab-1 is capable of both cell growth and cell the of The absence of in MAP kinase in and fibroblasts that IRS family members may not be for the activation of the kinase The role of Gab-1 in MAP kinase activation may depending on the cell Several have activation of the MAP kinases in several cell (18.Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (607) Google Scholar, 21.Takahashi-Tezuka M. Yoshida Y. Fukada T. Ohtani T. Yamanaka Y. Nishida K. Nakajima K. Hibi M. Hirano T. Mol. Cell. Biol. 1998; 18: 4109-4117Crossref PubMed Scopus (253) Google Scholar, S. M. V. J. W. Nature. 1996; PubMed Scopus Google Scholar). (18.Holgado-Madruga M. Emlet D.R. Moscatello D.K. Godwin A.K. Wong A.J. Nature. 1996; 379: 560-563Crossref PubMed Scopus (607) Google have that Gab-1 and SOS with Grb-2 that Gab-1 may with SOS for Grb-2 In this Gab-1 may the activation of the MAP kinase The absence of IGF-1-stimulated Gab-1 phosphorylation may this and for the activation of the MAP kinase the of overexpression of Gab-1 in our is not to with SOS for Grb-2 and in MAP kinase activation are In we have demonstrated that Gab-1 is not by the IGF-1 overexpression of Gab-1 in IRS-1-deficient cells can the IGF-1-stimulated expression of the immediate-early gene and markedly the ability of the cells to studies are to the mechanism by which Gab-1 in the of these biological end T. L. C. and J. for
Winnay et al. (Sat,) studied this question.