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Grb10 Inhibits Insulin-stimulated Insulin Receptor Substrate (IRS)-Phosphatidylinositol 3-Kinase/Akt Signaling Pathway by Disrupting the Association of IRS-1/IRS-2 with the Insulin Receptor*

Open AccessPublished:December 18, 2002DOI:https://doi.org/10.1074/jbc.M208518200
      Grb10 has been proposed to inhibit or activate insulin signaling, depending on cellular context. We have investigated the mechanism by which full-length hGrb10γ inhibits signaling through the insulin receptor substrate (IRS) proteins. Overexpression of hGrb10γ in CHO/IR cells and in differentiated adipocytes significantly reduced insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Inhibition occurred rapidly and was sustained for 60 min during insulin stimulation. In agreement with inhibited signaling through the IRS/PI 3-kinase pathway, we found hGrb10γ to both delay and reduce phosphorylation of Akt at Thr308 and Ser473 in response to insulin stimulation. Decreased phosphorylation of IRS-1/2 may arise from impaired catalytic activity of the receptor, since hGrb10γ directly associates with the IR kinase regulatory loop. However, yeast tri-hybrid studies indicated that full-length Grb10 blocks association between IRS proteins and IR, and that this requires the SH2 domain of Grb10. In cells, hGrb10γ inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in a dose-dependent manner, but did not affect IR catalytic activity toward Tyr972 in the juxtamembrane region and Tyr1158/1162/1163 in the regulatory domain. We conclude that binding of hGrb10γ to IR decreases signaling through the IRS/PI 3-kinase/AKT pathway by physically blocking IRS access to IR.
      IR
      insulin receptor
      BPS
      between the Pleckstrin and SH2
      CHO
      Chinese hamster ovary
      GFP
      green fluorescence protein
      IRS
      IR substrate
      PH
      Pleckstrin homology
      PI 3-kinase
      phosphatidylinositol 3-kinase
      RTK
      receptor-tyrosine kinase
      SH2
      Src-homology 2
      HA
      hemagglutinin
      aa
      amino acid
      The insulin receptor (IR)1 transmits signals through the actions of its intrinsic receptor tyrosine kinase. Ligand binding results in the autophosphorylation of the IR on multiple tyrosine residues (
      • White M.F.
      • Shoelson S.E.
      • Kentmann H.
      • Kahn C.R.
      ,
      • Hubbard S.R.
      • Lei W.
      • Ellis L.
      • Hendrickson W.A.
      ). Once phosphorylated, these residues serve as docking sites for specific intracellular effectors central to the transmission of the insulin signal. The insulin receptor substrate (IRS) family (IRS-1 through IRS-4) is one such group of proteins that recognizes the phosphorylated IR via their phosphotyrosine binding domains (PTB) (
      • Wolf G.
      • Trub T.
      • Ottinger E.
      • Groninga L.
      • Lynch A.
      • White M.F.
      • Miyazaki M.
      • Lee J.
      • Shoelson S.E.
      ,
      • Gustafson T.A.
      • He W.
      • Craparo A.
      • Schaub C.D.
      • O'Neill T.J.
      ). The IRS-PTB domain associates with the NPXY motif surrounding tyrosine 972 in the juxtamembrane region of the IR, which promotes the IR-IRS interaction (
      • Eck M.J.
      • Dhe-Paganon S.
      • Trub T.
      • Nolte R.T.
      • Shoelson S.E.
      ). Once in contact with the receptor, IRS is phosphorylated on multiple tyrosine residues by the IR tyrosine kinase. Numerous adaptor proteins and enzymes then associate with tyrosine-phosphorylated IRS via their Src homology 2 (SH2) domains and convey the insulin signal downstream (
      • Skolnik E.Y.
      • Lee C.H.
      • Batzer A.
      • Vicentini L.M.
      • Zhou M.
      • Daly R.
      • Myers M.J.J.
      • Backer J.M.
      • Ullrich A.
      • White M.F.
      ,
      • Sun X.J.
      • Crimmins D.L.
      • Myers M.G.J.
      • Miralpeix M.
      • White M.F.
      ).
      The insulin receptor-binding protein Grb10 is of considerable interest due to its potential to positively or negatively affect receptor-tyrosine kinase signaling. Grb10 is a member of a superfamily of adaptor proteins, which includes Grb7 and Grb14. This family of proteins shares several structural features, including a SH2 and a Pleckstrin homology (PH) domain (
      • Liu F.
      • Roth R.A.
      ). The C-terminal SH2 domain has been shown to associate with the IR (
      • He W.
      • Rose D.W.
      • Olefsky J.M.
      • Gustafson T.A.
      ,
      • Dong L.Q.
      • Farris S.
      • Christal J.
      • Liu F.
      ), and mutation of a critical arginine residue in this region of full-length Grb10 disrupts IR-Grb10 association in cells (
      • Langlais P.
      • Dong L.Q.
      • Hu D.
      • Liu F.
      ). A second domain termed the BPS (forbetween the Pleckstrin and SH2) has also been suggested as a second independent IR-interacting domain (
      • He W.
      • Rose D.W.
      • Olefsky J.M.
      • Gustafson T.A.
      ). Grb10 interacts with the regulatory kinase loop of the IR (
      • Dong L.Q.
      • Farris S.
      • Christal J.
      • Liu F.
      ,
      • Frantz J.D.
      • Giorgetti-Peraldi S.
      • Ottinger E.A.
      • Shoelson S.E.
      ,
      • O'Neill T.J.
      • Rose T.W.
      • Pillay T.S.
      • Hotta K.
      • Olefsky J.M.
      • Gustafson T.A.
      ), although studies have also reported a Grb10-interacting region in the IR C terminus (
      • Morrione A.
      • Valentinis B.
      • Li S.
      • Ooi J.Y.T.
      • Margolis B.
      • Baserga R.
      ,
      • Hansen H.
      • Svensson U.
      • Zhu J.
      • Laviola L.
      • Giorgino F.
      • Wolf G.
      • Smith R.J.
      • Riedel H.
      ). Several human Grb10 isoforms exist and most likely arise from alternative splicing. Two isoforms of hGrb10 differ in the PH domain. hGrb10γ encodes for the full-length protein, while Grb10α (previously named Grb-IR) lacks an intact PH domain as it contains a 46-amino acid deletion in this region (
      • Liu F.
      • Roth R.A.
      ,
      • Dong L.Q.
      • Du H.-Y.
      • Porter S.
      • Kolakowski J.L.F.
      • Lee A.V.
      • Mandarino L.J.
      • Fan J.B.
      • Yee D.
      • Liu F.
      ). A third isoform, hGrb10β is identical to hGrb10γ with the exception of 58 amino acids at the extreme N terminus of the γ isoform (
      • Frantz J.D.
      • Giorgetti-Peraldi S.
      • Ottinger E.A.
      • Shoelson S.E.
      ,
      • Dong L.Q.
      • Du H.-Y.
      • Porter S.
      • Kolakowski J.L.F.
      • Lee A.V.
      • Mandarino L.J.
      • Fan J.B.
      • Yee D.
      • Liu F.
      ).
      The functional role for Grb10 in insulin signaling remains controversial. Grb10 has been shown to positively stimulate insulin-induced mitogenesis (
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Swamy O.R.
      • Leone M.E.
      • Riedel H.
      ). Microinjection of a peptide fragment including the BPS and SH2 domain of Grb10 inhibited insulin-stimulated DNA synthesis (
      • O'Neill T.J.
      • Rose T.W.
      • Pillay T.S.
      • Hotta K.
      • Olefsky J.M.
      • Gustafson T.A.
      ,
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Swamy O.R.
      • Leone M.E.
      • Riedel H.
      ), which, if functioning as a dominant negative, is consistent with Grb10 as a positive regulator for cell growth. Several studies have indicated an inhibitory role for Grb10 as well. Overexpression of hGrb10α negatively regulates insulin receptor-mediated tyrosine phosphorylation of GTPase-activated protein (GAP)-associated protein p60 and IRS-1 (
      • Liu F.
      • Roth R.A.
      ). Furthermore, binding of Grb10 via its BPS/SH2 domains has been shown to inhibit IR catalytic activity in vitro (
      • Bereziat V.
      • Kasus-Jacobi A.
      • Perdereau D.
      • Cariou B.
      • Girard J.
      • Burnol A.F.
      ,
      • Stein E.G.
      • Gustafson T.A.
      • Hubbard S.R.
      ,
      • Mounier C.
      • Lavoie L.
      • Dumas V.
      • Mohammad-Ali K.
      • Wu J.
      • Nantel A.
      • Bergeron J.J.
      • Thomas D.Y.
      • Posner B.I.
      ).
      Stable expression of hGrb10α in CHO/IR reduces insulin-dependent phosphorylation of IRS-1 (
      • Liu F.
      • Roth R.A.
      ), although a direct effect of full-length Grb10 on IRS-1/2 and downstream effectors of the insulin signal, such as Akt, has not been reported. However, several studies indicate that Grb10 may regulate downstream events in various signaling pathways. Overexpression of hGrb10γ (Grb10ζ) in rat hepatocytes inhibits insulin-stimulated glycogen synthase activity, through a proposed novel pathway outside of the classical PI 3-kinase to Akt/glycogen synthase kinase-3 signaling (
      • Mounier C.
      • Lavoie L.
      • Dumas V.
      • Mohammad-Ali K.
      • Wu J.
      • Nantel A.
      • Bergeron J.J.
      • Thomas D.Y.
      • Posner B.I.
      ). Grb10 has also been found to associate with tyrosine-phosphorylated c-kit receptor, and synergistically promote Akt activation. This study indicated Grb10 forms a constitutive complex with Akt and proposed that Grb10 positively influenced Akt activity by promoting its translocation to the cell membrane leading to the phosphorylation and activation of this kinase (
      • Jahn T.
      • Seipel P.
      • Urschel S.
      • Peschel C.
      • Duyster J.
      ).
      Insulin-stimulated tyrosine phosphorylation of IRS proteins induces the association of the p85 regulatory subunit of PI 3-kinase and subsequently results in an increase in PI 3-kinase activity (
      • White M.F.
      ). As an IR-interacting protein, Grb10 has the potential to influence signaling through this IRS/PI 3-kinase pathway and moderate the actions of downstream effectors such as Akt. This study investigates the molecular mechanisms linked to the effects of Grb10 on insulin signaling. We find that hGrb10γ both delays and decreases Akt phosphorylation. This inhibition stems from a rapid and sustained inhibition of insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2. Our studies suggest that the mechanism for Grb10-mediated inhibition of insulin signaling results from a physical disruption of IRS association with phosphorylated residues of the IR kinase domain and/or the NPXY motif (Tyr972). The SH2 domain of Grb10 is essential for IR/IRS disruption. In addition, this region is required to facilitate Grb10 association with the IR and inhibition of IRS tyrosine phosphorylation in cells. This physical disruption of IR/IRS interaction by Grb10 functions to slow signaling relayed through tyrosine-phosphorylated IRS proteins.

      DISCUSSION

      Grb10 has been shown to play both a positive and negative role in insulin signaling, and these differences appear to depend both on cell type and on the Grb10 splice variant being studied. To understand these functional differences, we investigated the molecular mechanism of Grb10 action at the insulin receptor. Here, we show that full-length human Grb10 has an inhibitory effect on insulin-stimulated signaling through the IRS/PI 3-kinase pathway to Akt. Using a yeast tri-hybrid system, we found Grb10 can disrupt the association of IRS proteins with IR. The Grb10 SH2 domain is essential for this disruption and for complete inhibition of IRS tyrosine phosphorylation in cells. Our findings support a mechanism for Grb10-mediated inhibition of IRS/PI 3-kinase/Akt signaling, in which association of IRS proteins with the IR kinase domain and/or the juxtamembrane NPXY motif (Tyr972) is sterically hindered by Grb10.
      Previous studies have concluded that blocking IR catalytic activity by either full-length Grb10 or domain fragments results in decreased substrate phosphorylation (
      • Bereziat V.
      • Kasus-Jacobi A.
      • Perdereau D.
      • Cariou B.
      • Girard J.
      • Burnol A.F.
      ,
      • Stein E.G.
      • Gustafson T.A.
      • Hubbard S.R.
      ). In agreement with these studies, we have observed a Grb10-mediated decrease in phosphorylation of IR substrates, including IRS-1/2 (this study) (
      • Liu F.
      • Roth R.A.
      ), p62dok(
      • Liu F.
      • Roth R.A.
      ,
      • Wick M.J.
      • Dong L.Q.
      • Hu D.
      • Langlais P.
      • Liu F.
      ), and Shc (data not shown). However, Mournier et al.(
      • Mounier C.
      • Lavoie L.
      • Dumas V.
      • Mohammad-Ali K.
      • Wu J.
      • Nantel A.
      • Bergeron J.J.
      • Thomas D.Y.
      • Posner B.I.
      ) reported that overexpression of hGrb10 did not affect IRS tyrosine phosphorylation, PI 3-kinase activity or Akt activity in rat hepatocytes, although IR kinase activity and IR autophosphorylation were reportedly reduced. Our results in CHO/IR cells show that the catalytic activity of the RTK toward itself is not significantly impaired by Grb10. To a degree this is intuitive since tyrosine phosphorylation of the IR is necessary for Grb10 association. The rapid association of Grb10 with the IR could suggest that if Grb10 blocked the enzymatic activity of the RTK, autophosphorylation of the IR would be reduced in CHO/IR/hGrb10γ stable cells. However, we found that the overall tyrosine phosphorylation of the IR was also unaffected by the presence of hGrb10γ (data not shown). It is possible that the effects of Grb10 on catalytic activity arise from conformational changes that sterically hinder access of the kinase with its substrates. While we can not exclude inhibition of enzymatic activity of the insulin receptor for IRS-1/2 as a cause for decreased substrate phosphorylation, the observation that hGrb10 physically disrupts the interaction between IR and IRS proteins using the yeast tri-hybrid system supports structural interference as a mechanism for Grb10-mediated inhibition. The interaction of IRS proteins with the IR is transient in nature, and several negative feedback mechanisms exist in cells to prompt rapid dissociation of tyrosine-phosphorylated IRS proteins with the receptor (
      • Ravichandran L.V.
      • Esposito D.L.
      • Chen J.
      • Quon M.J.
      ,
      • Kawazoe Y.
      • Naka T.
      • Fujimoto M.
      • Kohzaki H.
      • Morita Y.
      • Narazaki M.
      • Okumura K.
      • Saitoh H.
      • Nakagawa R.
      • Uchiyama Y.
      • Akira S.
      • Kishimoto T.
      ,
      • Liu Y.F.
      • Paz K.
      • Herschkovitz A.
      • Alt A.
      • Tennenbaum T.
      • Sampson S.R.
      • Ohba M.
      • Kuroki T.
      • LeRoith D.
      • Zick Y.
      ). As a result, we were unable to detect an interaction between IR and IRS proteins by co-immunoprecipitation (data not shown), and cannot completely assess physical disruption as an alternative mechanism for Grb10-mediated inhibition in mammalian cells.
      Although both the BPS and SH2 domains can independently interact with the IR, the two regions together are necessary for disruption of interactions between IRS-1/2 and IR. Our results with the yeast tri-hybrid system indicate that the BPS domain with either an active or inactive SH2 domain is sufficient for disruption. This would suggest that the BPS domain mediates the interaction with the IR and the SH2 domain facilitates a physical disruption of IR/IRS interaction. When the Grb10 SH2 domain is deleted or replaced with the Shc SH2 domain, the IR/IRS interaction is not disrupted although both constructs interact with the IR in yeast two-hybrid assay. These findings support a structural role for the Grb10 SH2 domain in hindering IR/IRS interactions. However, Grb10 molecules lacking an intact SH2 domain cannot support complete inhibition of IRS tyrosine phosphorylation in mammalian cells (Fig. 7 B). Interestingly, while the BPS domain with either an active or inactive SH2 domain disrupted IR/IRS interactions in the yeast tri-hybrid studies, only Grb10 (full-length and BPS/SH2 fragment) with an active SH2 domain inhibited insulin-stimulated IRS-1 tyrosine phosphorylation in mammalian cells. Complete inhibition appeared to correlate with the ability of Grb10 to co-immunoprecipitate the IR. In contrast with results using yeast two-hybrid, in which the BPS domain is sufficient for interaction with the IR, regardless of an active SH2 domain (Ref.
      • He W.
      • Rose D.W.
      • Olefsky J.M.
      • Gustafson T.A.
      and Fig. 6), only Grb10 with an active SH2 domain could co-immunoprecipitate the IR in cells. The high sensitivity of the yeast system may allow for the detection of the BPS-mediated IR interaction and may override the need for an active SH2 domain. In mammalian cells, the SH2 domain may be needed to facilitate or strengthen BPS-mediated association with the IR. For this reason, we could not fully assess the structural requirement of the SH2 domain in physically blocking IRS-1/2 access to the IR in cells. In addition, we were unable to express fragments encoding only the BPS or SH2 domains, most likely due to their instability in mammalian cells. While we were unable to detect Grb10 association with the IR when the SH2 was inactivated or deleted, partial inhibition of IRS tyrosine phosphorylation with overexpression of these mutants suggests that some interaction via the BPS domain is likely.
      Full-length hGrb10γ significantly reduced phosphorylation of Akt on both Thr308 and Ser473 in CHO/IR cells. A similar inhibitory effect on phosphorylation of endogenous Akt was seen with adenoviral expression of hGrb10γ in differentiated adipocytes. Our findings differ from a recent study reporting mGrb10 functioned as a positive regulator of Akt in the c-kit signaling pathway by promoting relocalization to the membrane (
      • Jahn T.
      • Seipel P.
      • Urschel S.
      • Peschel C.
      • Duyster J.
      ). We do not find hGrb10 to co-immunoprecipitate with Akt in CHO/IR cells (data not shown). Protein interactions with Grb10 may be species-specific. Receptor-associated variations in recruited signaling pathways may also account for the observed differences in Grb10 function. IRS signaling is directly impaired by Grb10 interactions with IR and inhibition of Akt activity by hGrb10γ may stem from a specific requirement for IRS proteins and their supporting role in the activation of PI 3-kinase in response to insulin stimulation.
      In this study, the mechanism for Grb10-mediated inhibition of insulin signaling was investigated. We found that full-length hGrb10 could significantly inhibit insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-2 and subsequently delay signaling to the downstream effector, Akt. Autophosphorylation of the IR is not impaired by the binding of Grb10 to the receptor, which indicates functional activity of the RTK in cells. hGrb10γ inhibits insulin-stimulated tyrosine phosphorylation of IRS in a dose-dependent manner, and the interaction between IRS and the IR is disrupted when full-length Grb10 is used in the yeast tri-hybrid system. These studies describe a new molecular mechanism for Grb10-mediated inhibition. By structurally hindering access to the IR kinase domain and/or the NPXY motif (Tyr972), Grb10 inhibits tyrosine phosphorylation of IRS-1 and IRS-2 and functions to decrease intracellular insulin signaling through the IRS/PI 3-kinase pathway.

      Acknowledgments

      We thank Dr. Richard Roth for the Myc-tagged IRS-1 construct, Dr. Klein-Hitpass for the IRS-2 construct, Dr. Thomas A. Gustafson for the yeast strain GUSEGY48, pLexA IR pB42AD Grb10 constructs and pB42AD IRS-1/2 constructs, and Dr. Ben Margolis for the mouse Shc (p52) cDNA. We acknowledge Drs. Tong-Chuan He and Bert Vogelstein for the pAdEasy system.

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