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Growth Factor Receptor-binding Protein 10 (Grb10) as a Partner of Phosphatidylinositol 3-Kinase in Metabolic Insulin Action*

  • Youping Deng
    Footnotes
    Affiliations
    Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202
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  • Sujoy Bhattacharya
    Footnotes
    Affiliations
    Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202
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  • O.Rama Swamy
    Affiliations
    Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202
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  • Ruchi Tandon
    Affiliations
    Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202
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  • Yong Wang
    Affiliations
    Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202
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  • Robert Janda
    Affiliations
    Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202
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  • Heimo Riedel
    Correspondence
    Member, Barbara Ann Karmanos Cancer Institute. To whom correspondence should be addressed: Dept. of Biological Sciences, Wayne State University, Detroit, MI 48202-3917. Tel.: 313-577-7870 or 8338; Fax: 313-577-6891
    Affiliations
    Department of Biological Sciences, Wayne State University, Detroit, Michigan 48202
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  • Author Footnotes
    * This work was supported in part by the National Science Foundation Grant MCB-9808795, American Diabetes Association Grant 7-02-RA-76, National Institutes of Health Grant R01 CA77873, and Juvenile Diabetes Foundation International Grant 197048 (to H. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Present address: Division of Biology, Kansas State University, Manhattan, KS 66506-4901.
    § Present address: Dept. of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163.
Open AccessPublished:June 03, 2003DOI:https://doi.org/10.1074/jbc.M304599200
      The regulation of the metabolic insulin response by mouse growth factor receptor-binding protein 10 (Grb10) has been addressed in this report. We find mouse Grb10 to be a critical component of the insulin receptor (IR) signaling complex that provides a functional link between IR and p85 phosphatidylinositol (PI) 3-kinase and regulates PI 3-kinase activity. This regulatory mechanism parallels the established link between IR and p85 via insulin receptor substrate (IRS) proteins. A direct association was demonstrated between Grb10 and p85 but was not observed between Grb10 and IRS proteins. In addition, no effect of mouse Grb10 was observed on the association between IRS-1 and p85, on IRS-1-associated PI 3-kinase activity, or on insulin-mediated activation of IR or IRS proteins. A critical role of mouse Grb10 was observed in the regulation of PI 3-kinase activity and the resulting metabolic insulin response. Dominant-negative Grb10 domains, in particular the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed for glycogen synthesis, glucose and amino acid transport, and lipogenesis. In parallel, the same metabolic responses were substantially elevated by increased levels of Grb10. A similar role of Grb10 was confirmed in mouse L6 cells. In addition to the SH2 domain, the Pro-rich amino-terminal region of Grb10 was implicated in the regulation of PI 3-kinase catalytic activity. These regulatory roles of Grb10 were extended to specific insulin mediators downstream of PI 3-kinase including PKB/Akt, glycogen synthase kinase, and glycogen synthase. In contrast, a regulatory role of Grb10 in parallel insulin response pathways including p70 S6 kinase, ubiquitin ligase Cbl, or mitogen-activated protein kinase p38 was not observed. The dissection of the interaction of mouse Grb10 with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism.
      Grb10 was originally discovered as a partner of the epidermal growth factor (EGF)
      The abbreviations used are: EGF, epidermal growth factor; IR, insulin receptor; IRS-1/2, insulin receptor substrate-1/2; Grb10, growth factor receptor-binding protein 10; SH2, Src homology domain 2; PI 3-kinase, phosphatidylinositol 3-kinase; MAP kinase, mitogen-activated protein kinase; GSK3, glycogen synthase kinase 3; PKB/Akt, protein kinase B; HA, hemagglutinin; IP, immunoprecipitation; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PY, phosphotyrosine; IgG, immunoglobulin G; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; BPS, between the PH and SH2 domains; MOPS, 4-morpholinepropanesulfonic acid; ERK, extracellular signal-related kinase.
      1The abbreviations used are: EGF, epidermal growth factor; IR, insulin receptor; IRS-1/2, insulin receptor substrate-1/2; Grb10, growth factor receptor-binding protein 10; SH2, Src homology domain 2; PI 3-kinase, phosphatidylinositol 3-kinase; MAP kinase, mitogen-activated protein kinase; GSK3, glycogen synthase kinase 3; PKB/Akt, protein kinase B; HA, hemagglutinin; IP, immunoprecipitation; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; PY, phosphotyrosine; IgG, immunoglobulin G; PMSF, phenylmethylsulfonyl fluoride; BSA, bovine serum albumin; BPS, between the PH and SH2 domains; MOPS, 4-morpholinepropanesulfonic acid; ERK, extracellular signal-related kinase.
      receptor (
      • Ooi J.
      • Yajnik V.
      • Immanuel D.
      • Gordon M.
      • Moskow J.J.
      • Buchberg A.M.
      • Margolis B.
      ), however, a role in EGF action remains unsupported. Grb10 has been shown to associate with IR (
      • Liu F.
      • Roth R.A.
      ,
      • Hansen H.
      • Svensson U.
      • Zhu J.
      • Laviola L.
      • Giorgino F.
      • Wolf G.
      • Smith R.J.
      • Riedel H.
      ,
      • Frantz J.D.
      • Giorgetti-Peraldi S.
      • Ottinger E.A.
      • Shoelson S.E.
      ) and the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) (
      • Morrione A.
      • Valentinis B.
      • Li S.
      • Ooi J.Y.T.
      • Margolis B.
      • Baserga R.
      ,
      • Dey B.R.
      • Frick K.
      • Lopaczynski W.
      • Nissley S.P.
      • Furlanetto R.W.
      ,
      • Dong L.Q.
      • Farris S.
      • Christal J.
      • Liu F.
      ,
      • Dong L.Q.
      • Du H.
      • Porter S.G.
      • Kolakowski L.F.
      • Lee A.V.
      • Mandarino J.
      • Fan J.
      • Yee D.
      • Liu F.
      ) that carry out important metabolic and mitogenic functions, respectively. A preference was observed for IR in a direct comparison (
      • Laviola L.
      • Giorgino F.
      • Chow J.C.
      • Baquero J.A.
      • Hansen H.
      • Ooi J.
      • Zhu J.
      • Riedel H.
      • Smith R.J.
      ). At least seven splice variants have been identified in the human and mouse Grb10 gene, located on chromosomes 7 (human) or 11 (mouse) (
      • Jerome C.A.
      • Scherer S.W.
      • Tsui L.C.
      • Gietz R.D.
      • Triggs-Raine B.
      ,
      • Angrist M.
      • Bolk S.
      • Bently K.
      • Nallasamy S.
      • Halushka M.K.
      • Chakravarti A.
      ). This specific chromosomal location implicates a role of Grb10 in Silver-Russel syndrome (
      • Miyoshi N.
      • Kuroiwa Y.
      • Kohda T.
      • Shitara H.
      • Yonekawa H.
      • Kawabe T.
      • Hasegawa H.
      • Barton S.C.
      • Surani M.A.
      • Kaneko-Ishino T.
      • Ishino F.
      ,
      • Cattanach B.M.
      • Shibata H.
      • Hayashizaki Y.
      • Townsend K.M.S.
      • Ball S.
      • Beechey C.V.
      ), which remains to be demonstrated (
      • McCann J.A.
      • Zheng H.
      • Islam A.
      • Goodyer C.G.
      • Polychronakos C.
      ). Three mouse Grb10 variants have been reported, two of which have not been identified in humans (
      • Ooi J.
      • Yajnik V.
      • Immanuel D.
      • Gordon M.
      • Moskow J.J.
      • Buchberg A.M.
      • Margolis B.
      ,
      • Laviola L.
      • Giorgino F.
      • Chow J.C.
      • Baquero J.A.
      • Hansen H.
      • Ooi J.
      • Zhu J.
      • Riedel H.
      • Smith R.J.
      ). Four human sequences have originally been termed Grb-IR or hGrb10α, Grb10/IR-SV1, or hGrb-IRβ/Grb10, hGrb10γ and -δ (
      • Liu F.
      • Roth R.A.
      ,
      • Frantz J.D.
      • Giorgetti-Peraldi S.
      • Ottinger E.A.
      • Shoelson S.E.
      ,
      • Dong L.Q.
      • Farris S.
      • Christal J.
      • Liu F.
      ,
      • Dong L.Q.
      • Du H.
      • Porter S.G.
      • Kolakowski L.F.
      • Lee A.V.
      • Mandarino J.
      • Fan J.
      • Yee D.
      • Liu F.
      ,
      • O'Neill T.J.
      • Rose D.W.
      • Pillay T.S.
      • Hotta K.
      • Olefsky J.M.
      • Gustafson T.A.
      ) in the absence of a consistent nomenclature. A new consensus nomenclature has recently emerged (
      • Daly R.J.
      ,
      • Nantel A.
      • Mohammad-Ali K.
      • Sherk J.
      • Posner B.I.
      • Thomas D.Y.
      ) which has been used in this article.
      Based on structural similarities the Grb10 isoforms are members of the Grb7 superfamily of signaling mediators which include Grb7, Grb14, and the Caenorhabditis elegans MIG-10 and their splice variants (
      • Daly R.J.
      ,
      • Liu F.
      • Roth R.A.
      ,
      • Han D.C.
      • Shen T.L.
      • Guan J.L.
      ). Superfamily members share a Pro-rich putative SH3 domain binding region at the amino terminus, a region termed GM (Grb/Mig) that contains a Ras-associating-like domain (
      • Wojcik J.
      • Girault J.A.
      • Labesse G.
      • Chomilier J.
      • Mornon J.P.
      • Callebaut I.
      ) and is followed by a pleckstrin homology region, and a BPS (or IPS) domain located (between the PH and SH2 domains) (
      • He W.
      • Rose D.W.
      • Olefsky J.M.
      • Gustafson T.A.
      ,
      • Dong L.Q.
      • Porter S.
      • Hu D.
      • Liu F.
      ). All members carry an SH2 domain at the carboxyl terminus except MIG-10, which contains a Pro-rich region instead (
      • Manser J.
      • Roonprapunt C.
      • Margolis B.
      ). Both the SH2 and BPS domains have been implicated in the association with receptor tyrosine kinases including IR (
      • He W.
      • Rose D.W.
      • Olefsky J.M.
      • Gustafson T.A.
      ,
      • Dong L.Q.
      • Porter S.
      • Hu D.
      • Liu F.
      ,
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ). The family of Grb10 variants continues to grow (
      • Strausberg R.L.
      • Feingold E.A.
      • Grouse L.H.
      • Derge J.G.
      • Klausner R.D.
      • Collins F.S.
      • Wagner C.M.
      • Shenmen C.M.
      • Schuler G.D.
      • et al.
      ).
      Grb10 and its SH2 domain are dimeric in solution and the crystal structure of the Grb10 SH2 domain reveals a noncovalent dimeric conformation unique to the Grb7 family that will favor binding of dimeric, turn-containing phospho-Tyr sequences typical for IR and IGF-IR (
      • Stein E.G.
      • Ghirlando R.
      • Hubbard S.R.
      ). The Grb10 SH2 domain has been reported to associate, independently of phosphotyrosine, with Raf1 constitutively, and with MEK1 in response to insulin (
      • Nantel A.
      • Mohammad-Ali K.
      • Sherk J.
      • Posner B.I.
      • Thomas D.Y.
      ,
      • Nantel A.
      • Huber M.
      • Thomas D.Y.
      ). Basal phosphorylation on serine has been reported for Grb10, which was stimulated in response to EGF; similarly platelet-derived growth factor and fibroblast growth factor caused a mobility shift in the migration of Grb10 on SDS gels that was reversible by phosphatase treatment (
      • Ooi J.
      • Yajnik V.
      • Immanuel D.
      • Gordon M.
      • Moskow J.J.
      • Buchberg A.M.
      • Margolis B.
      ). Basal serine phosphorylation of one isoform was also stimulated by insulin, which was reversible by phosphatase, the MEK1 inhibitor PD98059, or the PI 3-kinase inhibitor wortmannin (
      • Dong L.Q.
      • Du H.
      • Porter S.G.
      • Kolakowski L.F.
      • Lee A.V.
      • Mandarino J.
      • Fan J.
      • Yee D.
      • Liu F.
      ). Tyrosine phosphorylation of Grb10 has been reported in response to insulin or vanadate that involves Src family kinases such as Src and Fyn (
      • Langlais P.
      • Dong L.Q.
      • Hu D.
      • Liu F.
      ). The underlying signaling mechanism may involve PI 3-kinase, p60 GAP-associated protein, the mitogen-activated protein kinase signaling cascade, and other pathways/mediators including vascular endothelial growth factor (
      • Giorgetti-Peraldi S.
      • Murdaca J.
      • Mas J.C.
      • Van Obberghen E.
      ), tyrosine kinases such as Jak2, Tec, and the oncogene Bcr-Abl (reviewed in Ref.
      • Riedel H.
      • Braun B.R.
      ). A role in mitogenesis has also been described for the related Grb7 and Grb14 both of which are also direct partners of IR (
      • Daly R.J.
      ,
      • Han D.C.
      • Shen T.L.
      • Guan J.L.
      ,
      • Kasus-Jacobi A.
      • Bereziat V.
      • Perdereau D.
      • Girard J.
      • Burnol A.F.
      ). This role also extends to metabolic insulin responses for Grb14 (
      • Kasus-Jacobi A.
      • Bereziat V.
      • Perdereau D.
      • Girard J.
      • Burnol A.F.
      ).
      Consequently, a role in the mitogenic response to insulin and other peptide hormones has been demonstrated for several Grb10 isoforms, however, a physiologic role in other insulin actions remained less defined (32 and reviewed in Ref.
      • Riedel H.
      • Braun B.R.
      ). The present study introduces mouse Grb10 as a critical component of the metabolic IR signaling complex that provides an alternative functional link between IR and p85 PI 3-kinase in response to insulin-stimulated metabolism.

      EXPERIMENTAL PROCEDURES

      All presented data are based on repeated experiments with the error shown between multiple measurements in bar graphs or with one representative experiment shown for immunoblots.
      Cell Culture—Mouse L6 cells, rat PC12 cells, and human IR overexpressing Rat1 cells (HIRcB) (
      • McClain D.A.
      • Maegawa H.
      • Levy J.
      • Huecksteadt T.
      • Dull T.J.
      • Lee J.
      • Ullrich A.
      • Olefsky J.M.
      ) were maintained in high glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1% (v/v) penicillin/streptomycin, in a 5% CO2 environment. Mouse 3T3-L1 cells were maintained in high glucose DMEM with 10% fetal bovine serum and 1% (v/v) penicillin/streptomycin, and were differentiated 2 days post-confluence by addition of 500 μm isobutylxanthine, 25 μm dexamethasone, and 4 μg/ml insulin (
      • Kayali A.G.
      • Eichhorn J.
      • Haruta T.
      • Morris A.J.
      • Nelson J.G.
      • Vollenweider P.
      • Olefsky J.M.
      • Webster N.J.G.
      ), and after 3 days cultured with the addition of only insulin. This medium was replaced every 3 days until cells were used after day 10 as well differentiated adipocytes in experiments. To elicit a metabolic response cells were subsequently typically stimulated with 100 nm insulin as specifically described for each procedure.
      Transient cDNA Transfection—Plasmid pHook2 (Invitrogen) carries a cytomegalovirus constitutive transcriptional promoter and served as a vector for transient expression of mouse Grb10δ (
      • Laviola L.
      • Giorgino F.
      • Chow J.C.
      • Baquero J.A.
      • Hansen H.
      • Ooi J.
      • Zhu J.
      • Riedel H.
      • Smith R.J.
      ). The complete protein coding cDNA of Grb10 had been removed from plasmid pRK5 (
      • Ooi J.
      • Yajnik V.
      • Immanuel D.
      • Gordon M.
      • Moskow J.J.
      • Buchberg A.M.
      • Margolis B.
      ) as a NruI-HindIII restriction fragment. Both sites were end-filled and inserted into the pHook2 expression plasmid (Invitrogen) at a unique SmaI restriction site to produce a mouse Grb10δ expression construct in pHook2. Subconfluent mouse L6 cells in 8-cm culture plates were rinsed with antibiotic-free medium before 3 ml of transfection mixture including 5–6 μg of Grb10 expression plasmid or the corresponding control plasmid, 20 μl of Plus reagent, and 30 μl of LipofectAMINE were added according to the instructions of the manufacturer (Invitrogen). Five hours later the transfection medium was replaced with complete culture medium supplemented with 10% fetal bovine serum. The effect of Grb10 expression (after cell starvation) was tested about 2 days posttransfection.
      Immunoprecipitation and Immunoblotting—Immunoprecipitation and immunoblotting were modified as described in Ref.
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      . Cells were cultured to quiescence for 20 h in serum-free DMEM supplemented with 0.1% BSA and stimulated with or without 100 nm insulin. Cells were rinsed twice with PBS and detergent cell extracts were prepared in ice-cold lysis buffer containing 50 mm HEPES, pH 7.4, 1% Triton X-100, 10% glycerol, 137 mm NaCl, 2 mm EDTA, 10 mm NaF, 100 mm Na3VO4, 10 mm sodium pyrophosphate, 10 μg/ml leupeptin, 10 μg/ml aprotinin, and 1 mm PMSF. Samples containing 300–500 μg of total protein were immunoprecipitated in a complex with protein A-Sepharose and antibody directed against Grb10 (Santa Cruz Biotechnology), p85 (Upstate Biotechnology or part of assay kit), IRS-1 (Upstate Biotechnology assay kit), IRS-2 (Upstate Biotechnology assay kit), IR (Upstate Biotechnology assay kit), or c-Cbl (Upstate Biotechnology assay kit). Precipitates were rinsed three times with lysis buffer, separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with specific antibodies as listed above or phospho-Tyr-specific antibody PY20 (Transduction Laboratories). For “far western” analysis (
      • Vidal M.
      • Goudreau N.
      • Cornille F.
      • Cussac D.
      • Gincel E.
      • Garbay C.
      ) the blot was first incubated with TAT-HA-Grb10 fusion protein followed by HA-specific antibody (Santa Cruz Biotechnology) and finally by goat anti-mouse IgG horseradish peroxidase as tertiary antibody (Santa Cruz Biotechnology). Proteins were visualized by the ECL system (Amersham Biosciences).
      Preparation of Cell Membrane-permeable Fusion Protein—The complete mouse Grb10δ protein-coding cDNA was introduced into Escherichia coli expression plasmid pTAT-HA (kindly provided by Steven F. Dowdy, Washington University School of Medicine) under control of the strong T7 phage transcriptional promoter (
      • Schwarze S.R.
      • Ho A.
      • Vocero-Akbani A.
      • Dowdy S.F.
      ). This plasmid carries amino-terminal coding sequences for a 6-amino acid His tag peptide to facilitate affinity purification of the recombinant protein and an 11-amino acid HIV TAT protein-derived peptide (YGRKKRRQRRR) that renders the resulting fusion proteins cell-membrane permeable. The complete protein coding region of mouse Grb10δ (
      • Laviola L.
      • Giorgino F.
      • Chow J.C.
      • Baquero J.A.
      • Hansen H.
      • Ooi J.
      • Zhu J.
      • Riedel H.
      • Smith R.J.
      ) was assembled starting just upstream of the ATG initiation codon of translation through an XhoI restriction site in the 3′-untranslated region. A 168-bp cDNA fragment encoding the 5′ end of the mouse Grb10δ protein coding sequence was amplified by PCR using primers (5′-GTCTTGGGGGTACCGGTGGTATGAACAACGATATTAACTCGTCC-3′) and (5′-ACGCCTGTGGCTGTCCCCGGGAGCTAGATG-3′) that introduced a 5′ KpnI site and 3′ XmaI site into the final PCR product. The remaining protein coding sequences of mouse Grb10δ (
      • Laviola L.
      • Giorgino F.
      • Chow J.C.
      • Baquero J.A.
      • Hansen H.
      • Ooi J.
      • Zhu J.
      • Riedel H.
      • Smith R.J.
      ) were retrieved as an XmaI-XhoI restriction fragment (
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ). Both fragments, which reconstituted the complete Grb10δ protein-coding region, were joined with a KpnI-XhoI restriction fragment of plasmid pTAT-HA to produce a cell membranepermeable TAT-HA-Grb10 fusion protein in E. coli.
      Recombinant plasmids were introduced into E. coli high level expression host BL21(DE3)LysS (Novagen). 500 ml of LB medium was inoculated with 100 ml of freshly saturated overnight culture and was induced with 500 μm isopropyl-β-thiogalactopyranoside at 37 °C to produce the fusion protein. After 7 h cells were sedimented and re-suspended in 8 m urea, 100 mm NaCl, 20 mm HEPES, pH 8.0. Cells were disrupted by sonication (Branson Sonifier-450, CT) on ice in a series of pulses for about 2 min until the solution became turbid and lysates were centrifuged at 15,000 × g for 15 min at 4 °C. Grb10 fusion protein was purified from the soluble fraction via the His tag by affinity chromatography on a nickel-nitrilotriacetic acid column (Qiagen) pre-equilibrated with 5 mm imidazole, 500 mm NaCl, 20 mm Tris, pH 8.0, 8 m urea. Fusion protein was eluted by stepwise addition of 5–10 ml of increasing concentrations of 100, 200, and 500 mm and 1 m imidazole in 8 m urea, 100 mm NaCl, 20 mm HEPES, pH 8.0. Fractions with an A 280 above 0.2 were pooled and initially dialyzed (Slide-A-Lyzer, Pierce) with a 3.5-kDa molecular mass cutoff for 2 h at 4 °C in PBS, pH 7.4, 4 m urea. Proteins were concentrated at 4 °C by centrifugation with Centriplus-3 (Millipore) at 3,000 × g at 3-kDa molecular mass cutoff. Samples were repeatedly reconstituted in PBS, pH 7.2, 10% glycerol to reach a peptide concentration of 0.2–0.3 mg/ml. Aliquots were shock frozen in liquid nitrogen and stored at –80 °C. Cell membrane-permeable fusion peptides representing the Grb10 amino-terminal Pro-rich region or SH2 domain had been prepared as described earlier fused with a sequence of the Drosophila melanogaster antennapedia homeoprotein (
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ). The SH2 domain had been expressed as a fusion peptide in E. coli and the Pro-rich region was represented by a synthetic peptide mimetic (synthesized by American Peptide Company). A synthetic peptide lacking Grb10 sequences or a dialyzed column eluate of a control E. coli cell extract served as peptide controls.
      Glycogen Synthesis—A protocol was followed as described below similar to the procedure outlined in Ref.
      • Wada T.
      • Sasaoka T.
      • Funaki M.
      • Hori H.
      • Murakami S.
      • Ishiki M.
      • Haruta T.
      • Asano T.
      • Ogawa W.
      • Ishihara H.
      • Kobayashi M.
      . Mouse L6 cells (after transfection) or 3T3-L1 adipocytes were seeded into 12-well plates at densities of 4–5 × 105 cells per well. Cells were cultured for 24 h and subsequently deprived of serum for 18–20 h in DMEM supplemented with 0.1% BSA. Cells were rinsed twice with ice-cold PBS followed by a 3-h incubation in 2.5 mm glucose, 0.1% BSA, 25 mm HEPES, pH 7.4. Cells were incubated for 30 min at 37 °C in the presence or absence of 100 nm insulin, cell membrane-permeable peptide mimetics (10 μg/ml), and/or 20 μm PI 3-kinase inhibitor LY 294002 and/or 20 μm p38 MAP kinase inhibitor SB203580. Subsequently, cells were incubated with 2.5–5.0 μl of d-[U-14C]glucose (200 μCi/ml, 2–4 mCi/mmol, Amersham Biosciences) for 1 h at 37 °C (final concentration 0.5–1 μCi/ml). Glycogen synthesis was terminated by rinsing cells three times with ice-cold PBS followed by cell lysis with 0.5 ml of 30% KOH for 1 h at 37 °C. Lysates were transferred to 1.5-ml microcentrifuge tubes, 50 μl of 20 mg/ml carrier glycogen (final concentration 2 mg/ml) was added, and cell lysates were incubated at 95 °C for 30 min. Samples were cooled to room temperature, 0.6 ml of ice-cold ethanol was added, and glycogen was precipitated for 16 h at –20 °C. Samples were sedimented at 3,000 × g for 10 min and the supernatant was aspirated. The precipitate was solubilized in 1 ml of H2O and incorporated radioactivity was determined by liquid scintillation spectroscopy.
      Glucose Transport—A protocol was followed as described below similar to the procedure outlined in Ref.
      • Wiese R.J.
      • Mastick C.C.
      • Lazar D.F.
      • Saltiel A.R.
      . 5 × 105 L6 cells (after transfection) or 1.5 × 105 3T3-L1 adipocytes per well (for 24-well plates) were starved for 3–5 h in serum-free DMEM supplemented with 0.1% BSA and cells were washed twice with KRPH buffer (1% bovine serum albumin, 1 mm MgSO4, 1 mm CaCl2, 136 mm NaCl, 5 mm Na2HPO4, 20 mm HEPES, pH 7.4). Cells were incubated for 15 min at 37 °C with 100 nm insulin and/or 10 μg/ml cell membrane-permeable peptide mimetics. 0.5 μl of 2-deoxy-[3H]glucose (1 mCi/ml, 10–20 Ci/mmol, Amersham Biosciences) was added for 4 more min (final concentration 0.5 μCi/ml) and nonspecific glucose transport was determined in the presence of 10 μm cytochalasin B, which was subtracted for final data presentation. Cells were subsequently rinsed twice with ice-cold PBS and lysed in 0.5 ml of 0.5 n NaOH, followed by neutralization with 0.5 ml of 2 m Tris, pH 6.8. Associated radioactivity was determined by liquid scintillation spectroscopy.
      Amino Acid Transport—A protocol was followed as described below similar to the procedure outlined in Ref.
      • McDowell H.E.
      • Eyers P.A.
      • Hundal H.S.
      . 5 × 105 L6 cells (after transfection) or 1.5 × 105 3T3-L1 adipocytes per well (in 24-well plates) were starved in serum-free DMEM supplemented with 0.1% BSA for 16 h. Cells were incubated for 1 h at 37 °C with 100 nm insulin and/or 10 μg/ml cell membrane-permeable peptide mimetics followed by the addition of 1 μl of 2-amino-[1-14C]isobutyric acid (50 μCi/ml, 50–62 mCi/mmol, Amersham Biosciences) for 10 min (final concentration 0.05 μCi/ml). Cells were rinsed three times with ice-cold PBS, lysed in 0.5 ml of 0.2 n NaOH for 30 min at 40 °C, and samples were neutralized with 0.5 ml of 0.2 n HCl. Associated radioactivity was determined by liquid scintillation spectroscopy.
      Lipogenesis—A protocol was followed as described below similar to the procedure outlined in Ref.
      • Wiese R.J.
      • Mastick C.C.
      • Lazar D.F.
      • Saltiel A.R.
      . 4–5 × 105 L6 cells (after transfection) or 3T3-L1 adipocytes per well (in 12-well plates) were starved for 18–20 h in serum-free medium supplemented with 0.1% BSA. Cells were incubated for 1 h at 37 °C with 100 nm insulin and/or 10 μg/ml cell membrane-permeable peptide mimetics in the presence of 0.5 μl of 2-deoxy-[3H]glucose (1 mCi/ml, 10–20 Ci/mmol, Amersham Biosciences) and 3.5 mm glucose (final concentration 0.5 μCi/ml). Cells were rinsed twice with ice-cold PBS, collected by scraping, and transferred to liquid scintillation vials. 5 ml of Toluene-Bray scintillation liquid was added and the mixture was incubated for 16 h at 25 °C. 4 ml of the organic phase was removed to quantify incorporated radioactivity by liquid scintillation spectroscopy.
      PI 3-Kinase Activity—A protocol was followed as described below similar to the procedure outlined in Refs.
      • Whitman M.
      • Kaplan D.R.
      • Schaffhausen B.
      • Cantley L.
      • Roberts T.M.
      and
      • Vlahos C.J.
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      . 106 differentiated 3T3-L1 adipocytes or IR overexpressing Rat1 (HIRcB) fibroblasts (
      • McClain D.A.
      • Maegawa H.
      • Levy J.
      • Huecksteadt T.
      • Dull T.J.
      • Lee J.
      • Ullrich A.
      • Olefsky J.M.
      ) in 5-cm culture plates were starved for 24 h in serum-free DMEM supplemented with 0.1% BSA. Cells were subsequently incubated with 10 μg/ml cell membrane-permeable peptides for 1 h at 37 °C before 100 nm insulin was added for 10 min. Detergent cell extracts were prepared in 1 ml of lysis buffer (1% Triton X-100, 10% glycerol (v/v), 137 mm NaCl, 2 mm EDTA, 10 mm NaF, 100 mm Na3VO4, 10 mm Na4P2O7, 10 μg/ml leupeptin, 1 mm PMSF, 50 mm HEPES, pH 7.4). For immunoprecipitation at 4 °C, 500 μg of total protein was incubated with p85 or IRS-1 antibody for 2 h and further for 1 h after addition of 25 μl of protein A-Sepharose. Immune complexes were collected by centrifugation at 4 °C and rinsed once with ice-cold lysis buffer and twice with 20 mm HEPES, pH 7.4, 150 mm NaCl, 1 mm Na3VO4, 10 μg/ml aprotinin, 1 mm PMSF. The sediment was re-suspended in 50 μl of kinase buffer (20 mm HEPES, pH 7.4, 1 mm EGTA, 10 mm MgCl2, 1 mm Na3VO4) including 0.2 mg/ml sonicated phosphatidylinositol (Sigma). 2–4 μl of [γ-32P]ATP (5 mCi/ml, 3000 Ci/mmol, PerkinElmer Life Sciences) was added to a final concentration of 60 nm. The kinase reaction was carried out for 20 min at 25 °C and terminated by adding 20 μl of 6 m HCl. Phosphatidylinositol was extracted with 200 μl of CHCl3/MeOH (1:1). The organic phase was washed with 80 μl of MeOH/HCl (1:1) and spotted on a Silica Gel 60 thin layer chromatography plate (Merck) pretreated with 1% potassium oxalate. Phospholipids were separated in running buffer containing CHCl3/MeOH/H2O2/NH4OH (75/100/25/15) and associated radioactivity was visualized by autoradiography.
      Akt/PKB Kinase Activity—Immunoprecipitation of Akt and analysis of its kinase activity was largely carried out according to the instructions in the employed experimental kit (Upstate Biotechnology). 106 differentiated 3T3-L1 adipocytes in 5-cm culture plates were starved for 20 h in serum-free DMEM. Cells were subsequently incubated with 10 μg/ml cell membrane-permeable peptides for 1 h at 37 °C before 100 nm insulin was added for 15 min. Cells were lysed in 1% Triton X-100, 10% glycerol (v/v), 50 mm HEPES, pH 7.6, 150 mm NaCl, 30 mm Na4P2O7, 10 mm NaF, 1 mm EDTA, 1 mm PMSF, 1 mm Na3VO4, 1 mm dithiothreitol, 1 μm Microcystin, and 10 μg/ml of each aprotinin, pepstatin, and leupeptin. 25 μl of protein A-Sepharose pre-equilibrated for 16 h at 4 °C and 4 μg of Akt-1/PKB pleckstrin homology domain antibody (part of the kit) were added to 1 ml of cell lysate and incubated under continuous suspension for 90 min at 4 °C. Immune complexes were precipitated and rinsed three times with 500 μl of lysis buffer containing 0.5 m NaCl, twice with 0.03% (w/v) Brij-35, 50 mm Tris-HCl, pH 7.5, 0.1 mm EGTA, 0.1% (v/v) 2-mercaptoethanol, and twice with 100 μl of AD buffer (100 mm MOPS, pH 7.2, 125 mm β-glycerol phosphate, 25 mm EGTA, 5 mm Na3VO4, 5 mm dithiothreitol). The immunoprecipitate was re-suspended in 10 μl of ice-cold AD buffer followed by the addition of 10 μl of Akt/GSK3 phosphorylation substrate peptide and 10 μl of protein kinase A inhibitor (part of the kit). Phosphorylation was carried out with 0.1 μCi/ml [γ-32P]ATP (5 mCi/ml, 3000 Ci/mmol, PerkinElmer Life Sciences) for 10 min at 37 °C under continuous suspension. The reaction was terminated by the addition of 20 μl of 40% trichloroacetic acid. After 5 min 40 μl of the reaction mixture was spotted on 2-cm2 P81 Whatman phosphocellulose paper, which was rinsed three times for 5 min each with 0.75% phosphoric acid and once with acetone. Associated radioactivity was determined by liquid scintillation spectroscopy.
      GSK3 Phosphorylation—106 differentiated 3T3-L1 adipocytes in 5-cm culture plates were cultured to quiescence for 20 h in serum-free DMEM. Cells were subsequently incubated with 10 μg/ml cell membrane-permeable peptides for 1 h and stimulated with 100 nm insulin for an additional 10 min at 37 °C. Cells were rinsed twice with ice-cold PBS and detergent cell extracts were prepared in ice-cold lysis buffer containing 50 mm HEPES, pH 7.4, 1% Triton X-100, 10% glycerol (v/v), 137 mm NaCl, 2 mm EDTA, 10 mm NaF, 100 mm Na3VO4, 10 mm Na4P2O7, 1 mm PMSF, and 10 μg/ml of each leupeptin and aprotinin. About 25 μg of cell protein extract (per lane) was separated by SDS-PAGE (8%), and proteins were transferred to a nitrocellulose membrane. Immunoblots with phospho-specific GSK3 antibody (Cell Signaling Technology) were visualized by the Amersham ECL system. p38 MAP Kinase Activity—p38 MAP kinase activity was largely assayed according to the instructions in the employed p38 MAP Kinase Assay Kit (Cell Signaling Technology). 106 differentiated 3T3-L1 adipocytes in 5-cm culture plates were starved for 20 h in serum-free DMEM. Cells were subsequently incubated with 10 μg/ml cell membrane-permeable peptides for 1 h and stimulated with 100 nm insulin for an additional 10 min at 37 °C. Cells were rinsed twice with ice-cold PBS and 500 μl of detergent cell extract (about 200 μg of total protein) was immunoprecipitated with immobilized phospho-p38 MAP kinase (Thr180/Tyr182) antibody for 16 h at 4 °C. Immunocomplexes were repeatedly rinsed, re-suspended in kinase buffer with 200 μm ATP and 2 μg of ATF-2 substrate, and incubated for 30 min at 30 °C. The reaction was terminated by adding 25 μl of SDS sample buffer and products were separated by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane and immunoblots with phospho-ATF-2 antibody were visualized by the Amersham ECL system.
      P70 S6 Kinase Activity—P70 S6 kinase activity was largely analyzed according to the instructions in the employed S6 kinase assay kit (Upstate Biotechnology). 106 differentiated 3T3-L1 adipocytes in 5-cm plates were starved for 20 h in serum-free medium. Cells were subsequently incubated with 10 μg/ml cell membrane-permeable peptides for 1 h and stimulated with 100 nm insulin for an additional 15 min at 37 °C. Cells were rinsed twice with ice-cold PBS and detergent cell extracts were prepared as described above for the Akt/PKB kinase activity assay. About 200 μg of total cellular protein was immunoprecipitated with p70 S6 kinase antibody (part of the kit) at 4 °C for 16 h. Immunocomplexes were rinsed and re-suspended in assay buffer with 10 μl of substrate peptide (AKRRRLSSLRA) and 5 μl of [γ-32P]ATP (5 mCi/ml, 3000 Ci/mmol, PerkinElmer Life Sciences, final concentration 0.3 μm). Reactions were carried out for 10 min at 30 °C. 25-μl aliquots were spotted on 2-cm2 P81 Whatman phosphocellulose paper, which was rinsed three times for 5 min each with 0.75% phosphoric acid and once with acetone. Associated radioactivity was determined by liquid scintillation spectroscopy.
      Glycogen Synthase Activity—A protocol was followed as described below similar to the procedure outlined in Ref.
      • Sharma P.M.
      • Egawa K.
      • Huang Y.
      • Martin J.L.
      • Huvar I.
      • Boss G.R.
      • Olefsky J.M.
      . 4 × 105 differentiated 3T3-L1 adipocytes per well in 12-well plates were starved for 4 h in serum-free DMEM lacking glucose and supplemented with 20 mm HEPES, pH 7.4, and 1% BSA. Cells were incubated with cell membrane-permeable peptides and insulin in KPRH buffer for 30 min as described above under “Glucose Transport.” Cells were rinsed twice with ice-cold PBS, collected by scraping, sonicated for 30 s on ice in 200 μl of homogenization buffer (50 mm Tris-HCl, pH 7.4, 10 mm EDTA, 10 mm NaF), and centrifuged at 10,000 × g for 10 min at 4 °C. 30 μl of homogenate (about 10 μg of protein) was added to 60 μl of glycogen synthase assay buffer containing 50 mm Tris-HCl, pH 7.4, 10 mm EDTA, 10 mm NaF, 10 mg/ml glycogen. Reactions were carried out in the presence of 5 μl of UDP-d-[U-14C]glucose (0.02 mCi/ml, 250–360 mCi/mmol, PerkinElmer Life Sciences) at a final concentration of 0.1 μCi/ml in the absence or presence of 2 mg/ml glucose 6-phosphate for 30 min at 30 °C. 45-μl aliquots were spotted on a Whatman No. 4 filter and dried. Filters were washed with ice-cold 70% ethanol once for 20 min at 4 °C, twice for 30 min at room temperature, once with acetone, and dried. UDP-[U-14C]glucose incorporation into glycogen was measured as the associated radioactivity by liquid scintillation spectroscopy.

      RESULTS

      Grb10 and Insulin-mediated Metabolic Responses in L6 Cells—To address the putative role of Grb10 in insulin metabolic action we started with an established physiologic response to insulin. We chose the stimulation of glycogen synthesis that can be observed as evidence for a metabolic response to insulin even in fibroblasts that are easily transfected by cDNA. Because fibroblasts lack the insulin-specific glucose transporter GLUT4, their glucose transport response is not representative of a physiologically significant metabolic response to insulin (
      • Mueckler M.
      ). Glycogen synthesis was investigated in response to Grb10 overexpression in mouse L6 cells in the presence and absence of insulin. We observed a 3-fold increase in response to insulin and a significant but smaller 2-fold increase in response to elevated levels of Grb10 in the absence of insulin (Fig. 1A). The combination of insulin and elevated levels of Grb10 showed a strongly synergistic effect, 6-fold elevated over basal non-stimulated levels, clearly indicating a stimulatory role of Grb10 in insulin-mediated glycogen synthesis. To test whether we could block insulin action by interfering with the function of normal cellular Grb10 we capitalized on peptide mimetics representing the SH2 domain or the amino-terminal Pro-rich region of Grb10 that we had earlier shown to act as dominant-negative forms of Grb10 (
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ). Both forms were used as cell membrane-permeable peptides fused to a transfer sequence of the Drosophila antennapedia homeodomain protein (
      • Prochiantz A.
      ) that had been effectively introduced into fibroblasts in our hands in previous experiments (
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ). Treatment of insulin-stimulated cells with either peptide abolished insulin stimulation of glycogen synthesis down to a basal level (Fig. 1A), suggesting that although the insulin response was blocked, other mechanisms were not affected. Next, we tested whether the dominant-negative cell membrane-permeable peptides would neutralize the effect of Grb10 cDNA overexpression. Both dominant-negative peptides eliminated the synergistic effect of the combination of increased Grb10 and insulin stimulation on glycogen synthesis but did not abolish the insulin-independent activity of Grb10 (Fig. 1A). This result implicates a critical role not only of Grb10, but specifically of its Pro-rich and SH2 domains in insulin-stimulated glycogen synthesis. This outcome is consistent with our earlier observations in insulin-stimulated mitogenesis, in particular DNA synthesis (
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ).
      Figure thumbnail gr1
      Fig. 1Opposing effects of elevated Grb10 and its dominant-negative domains on insulin-stimulated glycogen synthesis, glucose transport, lipogenesis, and amino acid transport in L6 cells. L6 cells were transfected with a Grb10 expression plasmid or with control plasmid (Grb10: +/–) and/or treated with cell membranepermeable peptide mimetics representing the Grb10 amino-terminal Pro-rich region (Pro), its SH2 domain (SH2), or control peptide (C). Cells were stimulated with insulin (+/–) and (A) the incorporation of d-[U-14C]glucose into glycogen was quantified after precipitation by liquid scintillation spectroscopy. B, the uptake of 2-deoxy-d-[3H]glucose or of 2-amino-[1-14C]isobutyric acid was quantified by liquid scintillation spectroscopy after cell lysis. Lipogenesis as measured by the incorporation of d-[U-14C]glucose was quantified by liquid scintillation spectroscopy of the organic phase. The average of several experiments each with multiple data points has been presented including the experimental error. C, cell membrane-permeable SH2 domain (SH2) and complete Grb10 (All) were purified from E. coli. Both constructs were similarly expressed in milligram amounts and purified after sonication in denaturing (8 m urea) conditions by nickel-nitriloacetate-agarose affinity chromatography. Both forms are shown on a Coomassie Brilliant Blue-stained SDS gel with relative size markers (lane M) for reference (90k and 16k are indicated in Da).
      To follow up on the observed Grb10-mediated regulation of glycogen synthesis, we investigated whether other typical insulin metabolic responses would be similarly stimulated by increased levels of Grb10. We tested the effect of Grb10 cDNA expression in mouse L6 cells on lipogenesis, amino acid, and glucose transport. For lipogenesis, amino acid, or glucose transport, insulin resulted in an ∼3-fold stimulation and elevated levels of Grb10 resulted in an ∼2-fold stimulation over basal levels (Fig. 1B). A synergistic response of up to 6-fold over basal levels was the result of combining insulin stimulation with increased levels of Grb10 (Fig. 1B). Our findings show a general stimulatory role of Grb10 in all tested physiologic responses to insulin. In combination with the reported interaction of Grb10 with IR (
      • Liu F.
      • Roth R.A.
      ,
      • Hansen H.
      • Svensson U.
      • Zhu J.
      • Laviola L.
      • Giorgino F.
      • Wolf G.
      • Smith R.J.
      • Riedel H.
      ), this suggests a role of Grb10 in the IR signaling complex in the regulation of a key switch in the insulin signal.
      Expression of Cell Membrane-permeable SH2 Domain and Complete Grb10 Peptide Mimetics—Experiments were based on cell membrane-permeable peptide mimetics of the Grb10 SH2 domain that had been expressed in E. coli and of the amino-terminal Pro-rich region that had been chemically synthesized. The SH2 domain had been fused to a motif of the Drosophila antennapedia protein, expressed in milligram amounts, purified by affinity chromatography, and is shown as a single protein band of about 16 kDa consistent with its predicted size (Fig. 1C). To evaluate the effect of increased levels of Grb10 in differentiated adipocytes we prepared complete cell membrane-permeable Grb10 proteins fused to HIV TAT sequences. This approach has been described to deliver large proteins across the cell membrane (
      • Schwarze S.R.
      • Ho A.
      • Vocero-Akbani A.
      • Dowdy S.F.
      ,
      • Schwarze S.R.
      • Hruska K.A.
      • Dowdy S.F.
      ). The complete mouse Grb10 fusion protein was similarly expressed in milligram amounts, purified by affinity chromatography, and is shown as a single protein band of about 90 kDa close to its predicted size (Fig. 1C). Both proteins were found to be physiologically active and cell membrane-permeable complete Grb10 fusion protein (Fig. 2) was found functionally indistinguishable from cDNA-expressed Grb10 (Fig. 1, A and B).
      Figure thumbnail gr2
      Fig. 2Opposing effects of elevated Grb10 and its dominant-negative domains on insulin-stimulated glycogen synthesis, glucose transport, lipogenesis, and amino acid transport in 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were either treated with cell membrane-permeable complete Grb10 protein (All) and/or cell membrane-permeable peptide mimetics representing the Grb10 amino-terminal Pro-rich region (Pro), its SH2 domain (SH2), or control peptide (C). Cells were stimulated with insulin (+/–) and (A) the incorporation of d-[U-14C]glucose into glycogen was quantified after precipitation by liquid scintillation spectroscopy. B, the uptake of 2-deoxy-d-[3H]glucose or (C) of 2-amino-[1-14C]isobutyric acid was quantified after cell lysis by liquid scintillation spectroscopy. D, lipogenesis as measured by the incorporation of d-[U-14C]glucose was quantified by liquid scintillation spectroscopy of the organic phase. The average of duplicate measurements is presented including the experimental error.
      Grb10 and Physiologic Insulin Responses in Differentiated 3T3-L1 Adipocytes—Cell membrane-permeable peptide mimetics representing complete Grb10, its SH2 domain, or its amino-terminal Pro-rich region were added to the culture medium of differentiated and starved mouse 3T3-L1 adipocytes in combination with insulin to evaluate their impact on insulin-stimulated physiologic responses. Initially, glycogen synthesis, glucose transport, amino acid transport, and lipogenesis were evaluated. In most physiologic assays, approximately a 4-fold (3- to 5-fold) stimulation was observed by insulin (Fig. 2). This response was largely abolished by treatment with either dominant-negative SH2 or Pro-rich peptide mimetic, typically less effectively with the Pro-rich domain. These findings indicate that the dominant-negative Grb10 domains block most of the metabolic insulin response, but did not display inhibition of the basal cellular activity, compatible with a specific role of Grb10 in insulin action. Introduction of complete cell membrane-permeable Grb10 increased the physiologic responses to insulin between 2- and 3-fold (Fig. 2), indistinguishable from the response observed in L6 cells after Grb10 overexpression from cDNA (Fig. 1, A and B). This increase is fully compatible with the observed inhibition by both domain-specific peptides assuming that both represent dominant-negative forms (
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ). The introduction of either one of the dominant-negative forms together with complete Grb10 effectively neutralized the insulin-stimulated Grb10 response but did not completely eliminate the basal cellular response to Grb10. Equal concentrations (10 μg/ml) of all peptides had been employed that resulted in higher numbers of molecules representing the Pro-rich region when compared with the SH2 domain and in particular when compared with complete Grb10. As a result, it is expected that both dominant-negative forms approximately neutralize the stimulatory effect of increased complete Grb10. However, the fact that the basal cellular response to Grb10 was not fully eliminated supports the specificity of the observed inhibition by both the SH2 and Pro-rich domains that appears to be limited to insulin-stimulated Grb10 functions. The fact that the inhibition observed for the Pro-rich region when compared with the SH2 domain was consistently less pronounced despite its significantly higher molar concentration indicates that the Pro-rich region only partially inhibits the insulin-stimulated Grb10 response (Fig. 2). This is plausible because other unidentified Grb10 domains will likely participate in the involved mechanism.
      Grb10 Regulation of Specific Signaling Mediators—To better define the signaling pathway in which Grb10 participates, we tested several representative mediators in differentiated 3T3-L1 adipocytes for their potential regulation by Grb10 including glycogen synthase, glycogen synthase kinase (GSK3), protein kinase B (PKB)/Akt, ubiquitin ligase c-Cbl, and p70 S6 kinase (
      • Zick Y.
      ). We started with components that have been directly implicated in the established metabolic response pathway to insulin. Glycogen synthase is directly involved in glycogen synthesis that we observed to be regulated by Grb10 (Figs. 1A and 2A). We observed a 3–4-fold stimulation of enzymatic activity in response to insulin that was essentially abolished by the Grb10 SH2 domain and partly by the Pro-rich region (Fig. 3A). The combination of increased levels of Grb10 and insulin resulted in stimulation of enzymatic activity to more than 10-fold over basal levels that was reduced to the level of the basal Grb10 response by simultaneous treatment with the Grb10 SH2 domain or Pro-rich region (Fig. 3A). We proceeded to investigate whether the observed stimulation of glycogen synthesis involved the activation of established upstream mediators in the implicated metabolic insulin signaling pathway. An established key pathway involves the activation of PI 3-kinase resulting in phospholipid ligands (phosphatidylinositol 3-phosphate) of phospholipid-dependent kinases PDK1 and PDK2 that activate PKB/Akt. This results in phosphorylation of GSK3 that stimulates glycogen synthase (
      • Kido Y.
      • Nakae J.
      • Accili D.
      ). We specifically tested GSK3 phosphorylation and PKB/Akt catalytic activity and found both responses regulated by Grb10 (Fig. 3, B and C). We observed a 4-fold stimulation of PKB/Akt catalytic activity in response to insulin that was mostly abolished by the Grb10 SH2 domain and partly by the Pro-rich region (Fig. 3B). The combination of increased levels of Grb10 and insulin resulted in stimulation of enzymatic activity to more than 6-fold over basal levels, which was essentially reduced to basal levels by simultaneous treatment with PI 3-kinase inhibitor LY294002 (Fig. 3B). This suggests that the role of Grb10 in this response is PI 3-kinase dependent. Activation of GSK3 was found similarly regulated by Grb10 when compared with PKB/Akt (Fig. 3C), compatible with the established sequential role of both components in the metabolic response pathway to insulin.
      Figure thumbnail gr3
      Fig. 3Effects of elevated Grb10 and its dominant-negative domains on known mediators of the insulin signaling response. Differentiated 3T3-L1 adipocytes were treated with cell membrane permeable complete Grb10 protein (All) and/or cell membrane-permeable peptide mimetics representing the Grb10 amino-terminal Pro-rich region (Pro), its SH2 domain (SH2), or control peptide (C). Cells were stimulated with insulin (+/–) and lysed. The average of duplicate measurements is presented for bar graphs including the experimental error. A, glycogen synthase activity was determined in a sonicated cell lysate in the presence and absence of glucose 6-phosphate by incorporation of UDP-d-[U-14C]glucose into glycogen, which was quantified by liquid scintillation spectroscopy. B, PKB/Akt catalytic activity and the effect of PI 3-kinase inhibitor (LY294002 (PI3K): +/–) were quantified by liquid scintillation spectroscopy after phosphorylation of an Akt-specific peptide substrate by [γ-32P]ATP. C, immunoblots from detergent cell lysates separated by SDS-PAGE are shown with a phospho-specific GSK3 antibody. D, phosphorylation of a p70 S6 kinase peptide substrate by [γ-32P]ATP was quantified by liquid scintillation spectroscopy. E, immunoprecipitate of c-Cbl (IP) from detergent cell lysates with c-Cbl antibody was separated by SDS-PAGE and analyzed by immunoblotting with phosphotyrosine-specific antibody (PY) or c-Cbl antibody. The positions of GSK3 and c-Cbl protein are indicated by arrows.
      In addition, we tested the potential impact of Grb10 on the activity of p70 S6 kinase (Fig. 3D) and on Tyr phosphorylation of the ubiquitin ligase c-Cbl (Fig. 3E), two components that participate in parallel insulin responses. In contrast to any of the earlier mentioned responses both activities remained completely unaffected by either increased levels of Grb10 or dominant-negative domains of Grb10 (Fig. 3, D and E). Because both components represent established insulin signaling mediators that showed the expected regulation by insulin in our experiments (Figs. 3, D and E), they appear to participate in parallel mechanisms that are independent of Grb10 (
      • Chang P.Y.
      • Le Marchand-Brustel Y.
      • Cheatham L.A.
      • Moller D.E.
      ,
      • Somwar R.
      • Sumitani S.
      • Taha C.
      • Sweeney G.
      • Klip A.
      ,
      • Baumann C.A.
      • Ribon V.
      • Kanzaki M.
      • Thurmond D.C.
      • Mora S.
      • Shigematsu S.
      • Bickel P.E.
      • Pessin J.E.
      • Saltiel A.R.
      ).
      Grb10 and the PI 3-Kinase Signaling Pathway—The inhibition of the Grb10-stimulated PKB/Akt catalytic activity that we observed in response to inhibitor LY294002 (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Yamamoto-Honda R.
      • Tobe K.
      • Kaburagi Y.
      • Ueki K.
      • Asai S.
      • Yachi M.
      • Shirouzu M.
      • Yodoi J.
      • Akanuma Y.
      • Yokoyama S.
      ) suggested a role of PI 3-kinase in Grb10 action (Fig. 3B). Similarly, we investigated whether Grb10 regulation of glycogen synthesis or glucose transport would be PI 3-kinase-dependent. For both responses an about 3-fold stimulation was observed for insulin that was significantly inhibited by the PI 3-kinase inhibitor LY294002 (Figs. 4, A and B). The combination of insulin and increased levels of Grb10 resulted in about a 5-fold stimulation over basal levels for both responses that was largely eliminated by LY294002 for glycogen synthesis and partially for glucose transport. In contrast, the glycogen synthesis response to insulin and to increased levels of Grb10 remained unaffected by the p38 MAP kinase inhibitor SB203580 (
      • Kramer R.M.
      • Roberts E.F.
      • Um S.L.
      • Borsch-Haubold A.G.
      • Watson S.P.
      • Fisher M.J.
      • Jakubowski J.A.
      ). Similarly, SB203580 did not affect Grb10 stimulation of glucose transport, whereas it partially inhibited the glucose transport response to insulin (Fig. 4, A and B). A potential regulatory role of Grb10 was also addressed directly on p38 MAP kinase catalytic activity by measuring phosphorylation of substrate ATF-2 (
      • Wang X.S.
      • Diener K.
      • Manthey C.L.
      • Wang S.
      • Rosenzweig B.
      • Bray J.
      • Delaney J.
      • Cole C.N.
      • Chan-Hui P.Y.
      • Mantlo N.
      • Lichenstein H.S.
      • Zukowski M.
      • Yao Z.
      ). The activity of p38 was found to be insulin responsive but no regulation was observed by the Grb10 SH2 domain, Pro-rich region, or by increased levels of complete Grb10 (Fig. 4C). These findings imply a role of PI 3-kinase but suggest against a role of p38 MAP kinase in the insulin response that is regulated by Grb10.
      Figure thumbnail gr4
      Fig. 4Effects of PI 3-kinase and p38 MAP kinase inhibitors and elevated Grb10 or its dominant-negative domains on insulin-stimulated metabolism and p38 MAP kinase catalytic activity. Differentiated 3T3-L1 adipocytes were either treated with cell membrane-permeable complete Grb10 protein (All) or control peptide (C) and/or PI 3-kinase inhibitor (LY 294002 (PI3K): +/–) or p38 MAP kinase inhibitor (SB 203580 (P38K): +/–). Cells were stimulated with insulin (+/–) and (A) glycogen synthesis was measured by the incorporation of d-[U-14C]glucose into glycogen and quantified after precipitation by liquid scintillation spectroscopy. B, uptake of 2-deoxy-d-[3H]glucose was quantified after cell lysis by liquid scintillation spectroscopy. The average of duplicate measurements is presented including the experimental error. C, immunoprecipitates with phospho-p38 antibody of detergent cell lysates were resolved by SDS-PAGE and analyzed by immunoblotting with a phosphorylation-specific antibody against p38 MAP kinase substrate ATF-2. The positions of ATF-2 protein and IgG are indicated by arrows.
      Grb10 and the Regulation of PI 3-Kinase Activity—To demonstrate a role of PI 3-kinase, a key switch in insulin action (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ), directly, we tested its total cellular activity in response to insulin stimulation in combination with dominant-negative Grb10 domains or increased levels of Grb10 in 3T3-L1 adipocytes. We confirmed substantial activation in response to insulin that was considerably reduced by the Grb10 SH2 domain or Pro-rich region and was significantly elevated by increased levels of Grb10 (Fig. 5A). This result points to a potential role of the Grb10 Pro-rich region in PI 3-kinase activation and confirms a role of the SH2 domain that may be in part explained by its involvement in the interaction of Grb10 with IR (
      • He W.
      • Rose D.W.
      • Olefsky J.M.
      • Gustafson T.A.
      ,
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ). To address whether Grb10 may affect PI 3-kinase activity by modulating the activity of other known regulators of p85 PI 3-kinase, we specifically tested the insulin receptor substrates IRS-1 and IRS-2 as well as IR itself. In contrast to total cellular PI 3-kinase activity, IRS-1-associated PI 3-kinase activity after co-precipitation with an IRS-1 antibody was found unaffected by increased levels of Grb10 (Fig. 5B). The activity of IRS-1 (Fig. 6A), IRS-2 (Fig. 6B), or IR (Fig. 6C) as measured by Tyr phosphorylation was found unaffected by the Grb10 SH2 domain, Pro-rich region, or by increased levels of Grb10. In these experiments the presence of all three mediators had been confirmed by immunoblotting with specific antibodies and Tyr phosphorylation of all three mediators was demonstrated in response to insulin stimulation (Fig. 6). Our observations indicate a role of Grb10 in the regulation of PI 3-kinase activity, however, without affecting the regulation of IRS proteins or any noticeable feedback on IR activity. This data suggests a regulatory role of Grb10 in PI 3-kinase activation via mechanisms that bypass and are in parallel to those mediated by IRS proteins.
      Figure thumbnail gr5
      Fig. 5Effects of elevated Grb10 and its dominant-negative domains on insulin-stimulated total PI 3-kinase activity and IRS-1-associated PI 3-kinase activity. Cells stimulated with insulin (+/–) were either treated with cell membrane-permeable complete Grb10 protein (All), cell membrane-permeable peptide mimetics representing the Grb10 amino-terminal Pro-rich region (Pro), its SH2 domain (SH2), control peptide (C), or with PI 3-kinase inhibitor (LY 294002: +/–). After immunoprecipitation of PI 3-kinase activity from detergent cell lysates, phosphorylation of phosphatidylinositol (PI) substrate in the presence of [γ-32P]ATP into phosphatidylinositol 3-phosphate (PI3P) was resolved by thin layer chromatography and is shown after autoradiography. A, total PI 3-kinase catalytic activity in differentiated 3T3-L1 adipocytes was immunoprecipitated with p85 antibody and analyzed. B, IRS-1-associated PI 3-kinase activity was immunoprecipitated (IP) with IRS-1 antibody (IRS-1) from IR overexpressing Rat1 cells (HIRcB) and analyzed. Blank immunoprecipitate (–) or immunoprecipitate with p85 antibody (p85) are shown as controls.
      Figure thumbnail gr6
      Fig. 6Effects of elevated Grb10 and its dominant-negative domains on insulin-stimulated IRS-1, IRS-2, and IR tyrosine phosphorylation. Differentiated 3T3-L1 adipocytes were stimulated with insulin (+/–) and were either treated with cell membrane-permeable complete Grb10 protein (All) or with cell membrane-permeable peptide mimetics representing the Grb10 amino-terminal Pro-rich region (Pro), its SH2 domain (SH2), or with control peptide (C). Tyr phosphorylation of IRS-1 (A), IRS-2 (B), or IR (C) was analyzed in immunoblots (Blot PY) with a phosphotyrosine-specific antibody after immunoprecipitation (IP) with specific antibodies for each of the three mediators from detergent cell lysates. The presence of each of the three mediators (identified by arrows) was confirmed in immunoblots with specific antibodies (Blot: IRS-1, IRS-2, and IR).
      Association of Grb10 with p85 PI 3-Kinase (Directly) and Other Components of the IR Signaling Complex—All observations were consistent with a molecular association of Grb10 with p85, the regulatory subunit of PI 3-kinase. This was tested by immunoprecipitating p85 with specific antibodies from NIH 3T3 fibroblasts overexpressing IR after insulin stimulation and analyzing the precipitate in immunoblots with Grb10 antiserum. A substantial association was observed in response to insulin indicating that Grb10 and p85 are direct or indirect cellular partners (Fig. 7A). This association was abolished when peptide mimetics of the Grb10 SH2 domain or Pro-rich region were added to the association reaction (Fig. 7A). This observation implicates a role of the tested Grb10 domains either directly in an association with p85 or in other interactions that are required for the interaction between Grb10 and p85. To begin to distinguish between these options and address whether the observed association was direct, immunoprecipitates with p85 antiserum were evaluated in a far western blot by overlay with glutathione S-transferase-Grb10 fusion protein (not shown) or HA-tagged complete Grb10 fusion protein (Fig. 7B). Grb10 was clearly found to bind to cellular p85 with both strategies and at a substantially increased level to p85 expressed from stably transfected cDNA (Fig. 7B). These observations indicate a direct and insulin-dependent association of Grb10 with p85 that correlates with the regulation of PI 3-kinase catalytic activity by Grb10. This direct association may represent a critical feature of the role of Grb10 in regulating the physiologic responses to insulin.
      Figure thumbnail gr7
      Fig. 7Insulin-dependent association of cellular Grb10 directly with p85 PI 3-kinase and indirectly with IRS-1 and IRS-2. A, Rat1 fibroblasts overexpressing IR (HIRcB) were stimulated with insulin (+/–). Detergent cell extracts were treated with cell membranepermeable peptide mimetics representing the Grb10 amino-terminal Pro-rich region (Pro), its SH2 domain (SH2), or with control peptide (C) and immunoprecipitated (IP) with p85 specific antibody. Immunoprecipitates were resolved by SDS-PAGE and analyzed by immunoblotting (Blot) with anti-Grb10-specific antiserum. B, untransfected PC12 cells (Transfection: –) or PC12 cells overexpressing p85 (Transfection: p85) were stimulated with insulin (+/–). Detergent cell lysates were immunoprecipitated with p85 antibody and proteins were separated by SDS-PAGE. Immunoblots were analyzed with p85 antibody (Blot p85) or by far western overlay with HA-tagged complete Grb10 followed by a HA tag-specific antibody (Overlay HA-Grb10). C, differentiated 3T3-L1 adipocytes were stimulated with insulin (+/–). Detergent cell lysates were immediately separated by SDS-PAGE (IP: –) or were immunoprecipitated with Grb10-specific (Grb10), IRS-1-specific (IRS-1), or IRS-2-specific (IRS-2) antibody prior to SDS-PAGE. Proteins were identified by immunoblotting with specific antibody directed against IRS-1 or IRS-2, respectively (Blot IRS), or by far western overlay with HA-tagged complete Grb10 followed by HA tag-specific antibody (Overlay HA-Grb10). D, Rat1 fibroblasts overexpressing IR (HIRcB) were stimulated with insulin (+/–). Detergent cell extracts were treated with cell membrane-permeable complete Grb10 (All), control peptide (C), or cell membrane-permeable peptide mimetics representing the Grb10 amino-terminal Pro-rich region (Pro) or its SH2 domain (SH2). Immunoprecipitates with IRS-1 or p85 antibodies were separated by SDS-PAGE and analyzed by immunoblotting (Blot p85) with p85 antibody. Specific mediators have been identified by arrows on the right.
      To begin to elucidate the functional relationship between Grb10 and IRS proteins, we investigated whether an association could be demonstrated by co-immunoprecipitation. In response to insulin stimulation both IRS-1 and IRS-2 were identified with specific antibodies in immunoblots after immunoprecipitation of detergent cell lysates with Grb10 antibody (Fig. 7C). To investigate whether a direct association with Grb10 could be demonstrated, we tested IRS-1 or IRS-2 immunoprecipitates with HA-tagged complete Grb10 by far western analysis in an overlay assay. In contrast to the direct association that we observed between p85 and Grb10 (Fig. 7B) we were unable to demonstrate a direct association between IRS-1 or IRS-2 and Grb10 (Fig. 7C). In this context the presence of activated IRS protein had been demonstrated in parallel control experiments (see also Fig. 6, A and B). The observed indirect association between IRS-1 or IRS-2 and Grb10 can be explained via IR or p85 both of which associate with either type of mediator (
      • Liu F.
      • Roth R.A.
      ,
      • Hansen H.
      • Svensson U.
      • Zhu J.
      • Laviola L.
      • Giorgino F.
      • Wolf G.
      • Smith R.J.
      • Riedel H.
      ,
      • Myers M.G.J.
      • White M.F.
      ,
      • Van Horn D.J.
      • Myers Jr., M.G.
      • Backer J.M.
      ,
      • White M.F.
      ). Finally, we investigated the potential impact of Grb10 on the association between IRS-1 and p85. In these experiments immunocomplexes formed in the presence or absence of dominant-negative Grb10 peptides or increased levels of Grb10 were precipitated from cell lysates with IRS-1 antibody and the associated p85 was demonstrated by immunoblotting (Fig. 7D). No impact was observed on the insulin-mediated association between IRS-1 and p85 by any of the tested forms of Grb10. In combination, our results suggest that Grb10 regulates PI 3-kinase activity by directly associating with p85 in a parallel mechanism to the action of IRS-1 or IRS-2, which independently couple IR to p85.

      DISCUSSION

      In this study we evaluated the role of mouse Grb10 (
      • Ooi J.
      • Yajnik V.
      • Immanuel D.
      • Gordon M.
      • Moskow J.J.
      • Buchberg A.M.
      • Margolis B.
      ,
      • Laviola L.
      • Giorgino F.
      • Chow J.C.
      • Baquero J.A.
      • Hansen H.
      • Ooi J.
      • Zhu J.
      • Riedel H.
      • Smith R.J.
      ) in insulin metabolic action in mouse cells to eliminate potential inter-specific functional differences. Our results are based on two complementary major experimental strategies, introduction of dominant-negative peptide mimetics representing specific Grb10 domains and elevation of the cellular level of complete Grb10. In the first approach we introduced peptide mimetics of the Grb10 SH2 domain or the amino-terminal Pro-rich region that had been rendered cell membrane-permeable by attaching a 16-amino acid fragment of the Drosophila antennapedia homeodomain protein as a transfer sequence. We have earlier shown both Grb10 fusion peptides to act dominant-negatively and block the function of cellular Grb10 (
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ). In the second strategy we increased the cellular level of complete Grb10 by expression from cDNA. Alternatively, we introduced complete, cell membrane-permeable Grb10 protein fused to an HIV TAT transfer sequence. The cell membrane-permeable forms of Grb10 allowed us to employ differentiated 3T3-L1 adipocytes as an experimental model that is resistant to cDNA transfection but provides a meaningful physiologic insulin response. Experimental results were indistinguishable between mouse L6 cells that had been transfected with complete Grb10 cDNA (Fig. 1) and differentiated mouse 3T3-L1 adipocytes that had been subjected to complete cell membrane-permeable Grb10 (Fig. 2). The efficiency of cell membrane-permeable peptide delivery has been described to approach 100% of exposed cells (
      • Schwarze S.R.
      • Hruska K.A.
      • Dowdy S.F.
      ,
      • Robbins P.B.
      • Oliver S.F.
      • Sheu S.M.
      • Goodnough J.B.
      • Wender P.
      • Khavari P.A.
      ). It has routinely been found very high, exceeding the efficiencies in our hands that are typically observed by cDNA transfection (
      • Riedel H.
      • Yousaf N.
      • Zhao Y.
      • Dai H.
      • Deng Y.
      • Wang J.
      ). This is consistent with the efficiency by which in particular the Grb10 SH2 domain peptide blocks any insulin response tested in this study (Figs. 2 and 3). The observed block of the response by 90% or more requires that more than 90% of cells are reached by the peptides.
      We tested several metabolic responses to insulin including glycogen synthesis, glucose and amino acid transport, and lipogenesis. Increased levels of Grb10 were found to stimulate each response. When combined with insulin, substantial synergism was observed that resulted in severalfold higher stimulation than the normal insulin response (Figs. 1 and 2). Both individually tested Grb10 domain peptide mimetics substantially inhibited any tested metabolic response. The inhibition observed with the SH2 domain peptide frequently completely eliminated the observed insulin response and routinely exceeded the effect observed with the Pro-rich region (Figs. 1 and 2). A similar pattern had been demonstrated for the mitogenic insulin response and may be explained by the importance of the SH2 domain in the association of Grb10 with IR (
      • He W.
      • Rose D.W.
      • Olefsky J.M.
      • Gustafson T.A.
      ,
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ) and with other mediators, whereas the role of the Pro-rich region has not yet been elucidated.
      The effective block of insulin-stimulated glycogen synthesis by the Grb10 SH2 and Pro-rich peptide mimetics (Figs. 1A and 2) indicated effective delivery of the peptides into cells and the importance of Grb10 function in this metabolic response. In the presence of increased levels of Grb10 by cDNA transfection or peptide delivery of complete Grb10, again most of the insulin response was blocked (within the indicated experimental error) by the addition of dominant-negative peptide mimetics. However, the signal remained near the level reached by Grb10 in the absence of insulin (Figs. 1A and 2). These findings suggest that the observed Grb10 activity also carries an insulin-independent component that is not suppressed by either dominant-negative peptide. Consequently, the inhibitory action of the peptides is in part compensated by increased levels of Grb10 and appears specific to the Grb10-mediated insulin response rather than as an unspecific inhibitory activity of a general metabolic response. These results implicate a critical role not only of Grb10 but specifically of its Pro-rich region and SH2 domain in insulin-stimulated metabolic actions, similar to what the authors earlier observed in insulin-stimulated mitogenesis including DNA synthesis (
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ).
      The functional specificity of the dominant-negative peptide mimetics is further supported by two observations. First, no impact of any peptide mimetic was observed on the mitogenic response to EGF (in which Grb10 has not been implicated) and second, no impact of the Pro-rich peptide (in contrast to the SH2 domain) was observed on the mitogenic response to platelet-derived growth factor (
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ). Finally, the observation that both dominant-negative Grb10 peptide mimetics largely neutralized the metabolic insulin response but did not eliminate all basal Grb10 activity (Figs. 1A and 2) suggests their specific action in the insulin signaling pathway rather than a broad inhibition of basal metabolic activity.
      A key pathway that has been critically implicated in the metabolic responses to insulin involves Tyr phosphorylation of IRS proteins (by activated IR) that associate with the regulatory subunit p85 and lead to catalytic activation of PI 3-kinase (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). PI 3-kinase activation generates phospholipid ligands (phosphatidylinositol 3-phosphate) of phospholipid-dependent kinases PDK1 and PDK2 that activate PKB/Akt. This leads to phosphorylation of GSK3 that in turn stimulates glycogen synthase activity (
      • Zick Y.
      ,
      • Kido Y.
      • Nakae J.
      • Accili D.
      ). In addition to glycogen synthesis, we specifically tested glycogen synthase activity, GSK3 phosphorylation, and PKB/Akt catalytic activity and found all responses to be regulated by Grb10 (Fig. 3, A–C). A role of Grb10 in a PI 3-kinase-dependent mechanism was implicated by the observed sensitivity to the PI 3-kinase inhibitor LY294002 (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Yamamoto-Honda R.
      • Tobe K.
      • Kaburagi Y.
      • Ueki K.
      • Asai S.
      • Yachi M.
      • Shirouzu M.
      • Yodoi J.
      • Akanuma Y.
      • Yokoyama S.
      ), which eliminated insulin and Grb10 regulation of PKB/Akt activity (Fig. 3B) and of glycogen synthesis (Fig. 4A) but only partially of glucose transport (Fig. 4B). This observation suggests that insulin-mediated glycogen synthesis is substantially dependent on Grb10-mediated PI 3-kinase activation, whereas the regulation of insulin-mediated glucose transport by Grb10 appears to involve additional, PI 3-kinase-independent mechanisms that have been reported (
      • Baumann C.A.
      • Ribon V.
      • Kanzaki M.
      • Thurmond D.C.
      • Mora S.
      • Shigematsu S.
      • Bickel P.E.
      • Pessin J.E.
      • Saltiel A.R.
      ,
      • Isakoff S.J.
      • Taha C.
      • Rose E.
      • Marcusohn J.
      • Klip A.
      • Skolnik E.Y.
      ).
      Insulin-stimulated glucose transport involves parallel signaling mechanisms. Some involve p38 MAP kinase, whereas we found alternative Grb10-mediated mechanisms to be p38 MAP kinase-independent (
      • Moxham C.M.
      • Tabrizchi A.
      • Davis R.J.
      • Malbon C.C.
      ,
      • Sweeney G.
      • Somwar R.
      • Ramlal T.
      • Volchuk A.
      • Ueyama A.
      • Klip A.
      ,
      • Blair A.S.
      • Hajduch E.
      • Litherland G.J.
      • Hundal H.S.
      ,
      • Somwar R.
      • Kim D.Y.
      • Sweeney G.
      • Huang C.
      • Niu W.
      • Lador C.
      • Ramlal T.
      • Klip A.
      ). This view is supported by our experiments involving the p38 MAP kinase inhibitor SB203580 (Fig. 4, A and B) as well as p38 MAP kinase substrate ATF-2 (Fig. 4C). A significant impact of p38 MAP kinase inhibitor SB203580 was not observed on Grb10-stimulated glycogen synthesis or glucose transport, or on insulin-stimulated glycogen synthesis, whereas insulin-stimulated glucose transport was partially affected (Fig. 4, A and B) (
      • Sweeney G.
      • Somwar R.
      • Ramlal T.
      • Volchuk A.
      • Ueyama A.
      • Klip A.
      ,
      • Somwar R.
      • Kim D.Y.
      • Sweeney G.
      • Huang C.
      • Niu W.
      • Lador C.
      • Ramlal T.
      • Klip A.
      ). Similarly, no significant changes in p38 MAP kinase catalytic activity were observed in response to any Grb10 peptide mimetic (Fig. 4C). Overall, our data consistently did not observe any role of Grb10 in the regulation of p38 MAP kinase function. It is currently unclear whether Grb10 may activate signaling mechanisms downstream of p38 MAP kinase and the (predicted) sensitivity of ATF-2 phosphorylation to SB203580 (
      • Farassati F.
      • Yang A.D.
      • Lee P.W.
      • Patrick W.K.
      ) has not been formally confirmed. In addition, key mediators in alternative insulin-responsive signaling pathways including p70 S6 kinase and ubiquitin ligase c-Cbl (
      • Chang P.Y.
      • Le Marchand-Brustel Y.
      • Cheatham L.A.
      • Moller D.E.
      ,
      • Somwar R.
      • Sumitani S.
      • Taha C.
      • Sweeney G.
      • Klip A.
      ,
      • Baumann C.A.
      • Ribon V.
      • Kanzaki M.
      • Thurmond D.C.
      • Mora S.
      • Shigematsu S.
      • Bickel P.E.
      • Pessin J.E.
      • Saltiel A.R.
      ) remained unaffected by Grb10 regulation as shown in the presence of dominant-negative peptides or increased levels of Grb10 (Fig. 3, D and E). In combination, these results point to a specific role of Grb10 in the regulation of metabolic insulin responses through the PI 3-kinase pathway when compared with alternative insulinresponsive mechanisms.
      Regulation of total cellular and insulin-mediated PI 3-kinase catalytic activity was directly demonstrated in the presence of increased levels of Grb10 or dominant-negative peptide mimetics (Fig. 5A). However, IRS-I-associated PI 3-kinase activity as demonstrated by co-immunoprecipitation with IRS-I-specific antibody was not found to be regulated by Grb10 and while insulin-dependent, remained unchanged by increased levels of Grb10 (Fig. 5B). Key aspects of the regulation of PI 3-kinase activity in response to insulin have been well described and involve the activation of IR including its autophosphorylation on Tyr, the Tyr phosphorylation of IRS proteins by IR, and the resulting association of p85 with IRS proteins (
      • Shepherd P.R.
      • Withers D.J.
      • Siddle K.
      ). To begin to address the role of Grb10 in this complex we tested whether Grb10 would associate with any of the signaling mediators mentioned above or regulate their phosphorylation. Experiments involving immunoprecipitation with specific antibodies against any mediator followed by immunoblotting with phospho-Tyr-specific antibody showed that increased levels of Grb10 or individual dominant-negative Grb10 domains did not affect insulin-stimulated Tyr phosphorylation of IR, IRS-1, or IRS-2 (Fig. 6). A direct association between IR and Grb10 (
      • Liu F.
      • Roth R.A.
      ,
      • Hansen H.
      • Svensson U.
      • Zhu J.
      • Laviola L.
      • Giorgino F.
      • Wolf G.
      • Smith R.J.
      • Riedel H.
      ) as well as between IR and IRS proteins (
      • Myers M.G.J.
      • White M.F.
      ,
      • White M.F.
      ) or directly between IR and p85 (
      • Van Horn D.J.
      • Myers Jr., M.G.
      • Backer J.M.
      ) had been established earlier that implicates all components in the same signaling complex. The insulin-dependent association between p85 and Grb10 was demonstrated in co-immunoprecipitation experiments that involved the Grb10 SH2 domain and Pro-rich region because both respective peptide mimetics blocked the association with p85 (Fig. 7A). Immunoprecipitated p85 PI 3-kinase was recognized in overlay assays with HA-tagged complete Grb10 protein demonstrating a direct interaction between both mediators (Fig. 7B). Overlay assays to evaluate a direct association of IRS proteins in immunoblots with HA-tagged complete Grb10 protein were negative, however, an insulin-dependent association between IRS proteins and Grb10 was demonstrated in co-immunoprecipitation experiments (Fig. 7C). Our data suggest that this association is indirect presumably involving IR and possibly p85 both of which directly associate with IRS proteins as well as with Grb10 (
      • Liu F.
      • Roth R.A.
      ,
      • Hansen H.
      • Svensson U.
      • Zhu J.
      • Laviola L.
      • Giorgino F.
      • Wolf G.
      • Smith R.J.
      • Riedel H.
      ,
      • Myers M.G.J.
      • White M.F.
      ,
      • Van Horn D.J.
      • Myers Jr., M.G.
      • Backer J.M.
      ,
      • White M.F.
      ). The established insulin-dependent association between p85 and IRS-I was demonstrated in co-immunoprecipitation experiments and was not found regulated by Grb10 when exposed to either elevated levels of Grb10 or dominant-negative Grb10 peptide mimetics (Fig. 7D). No evidence was observed for any scavenging of p85 away from IRS-1.
      The interaction of Grb10 with IR and IGF-IR has been addressed in a number of independent studies. Different approaches have implicated different receptor sites in the association with Grb10 and the underlying mechanism is still being unraveled (
      • Hansen H.
      • Svensson U.
      • Zhu J.
      • Laviola L.
      • Giorgino F.
      • Wolf G.
      • Smith R.J.
      • Riedel H.
      ,
      • Frantz J.D.
      • Giorgetti-Peraldi S.
      • Ottinger E.A.
      • Shoelson S.E.
      ,
      • Morrione A.
      • Valentinis B.
      • Li S.
      • Ooi J.Y.T.
      • Margolis B.
      • Baserga R.
      ,
      • Dey B.R.
      • Frick K.
      • Lopaczynski W.
      • Nissley S.P.
      • Furlanetto R.W.
      ,
      • Dong L.Q.
      • Farris S.
      • Christal J.
      • Liu F.
      ,
      • Dong L.Q.
      • Du H.
      • Porter S.G.
      • Kolakowski L.F.
      • Lee A.V.
      • Mandarino J.
      • Fan J.
      • Yee D.
      • Liu F.
      ,
      • Laviola L.
      • Giorgino F.
      • Chow J.C.
      • Baquero J.A.
      • Hansen H.
      • Ooi J.
      • Zhu J.
      • Riedel H.
      • Smith R.J.
      ,
      • O'Neill T.J.
      • Rose D.W.
      • Pillay T.S.
      • Hotta K.
      • Olefsky J.M.
      • Gustafson T.A.
      ,
      • Nantel A.
      • Mohammad-Ali K.
      • Sherk J.
      • Posner B.I.
      • Thomas D.Y.
      ,
      • Dong L.Q.
      • Porter S.
      • Hu D.
      • Liu F.
      ,
      • Stein E.G.
      • Ghirlando R.
      • Hubbard S.R.
      ,
      • Stein E.G.
      • Gustafson T.A.
      • Hubbard S.R.
      ). A role of Grb10 has been documented in insulin and IGF-I action mostly at the level of mitogenesis with a number of distinct experimental strategies (
      • O'Neill T.J.
      • Rose D.W.
      • Pillay T.S.
      • Hotta K.
      • Olefsky J.M.
      • Gustafson T.A.
      ,
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ,
      • Riedel H.
      • Braun B.R.
      ,
      • Morrione A.
      • Valentinis B.
      • Resnicoff M.
      • Xu S.Q.
      • Baserga R.
      ). Some observations were found to vary with the specific cellular background including the specific Grb10 variants present in a given specific cell type (reviewed in Refs.
      • Riedel H.
      • Braun B.R.
      and
      • Morrione A.
      ). Initial observations reported reduced insulin-dependent Tyr phosphorylation of IRS-1, GAP-associated protein p60, and diminished activation of PI 3-kinase in response to human Grb10β overexpression in Chinese hamster ovary cells (
      • Liu F.
      • Roth R.A.
      ). Overexpression of the related mediator Grb14 in Chinese hamster ovary cells inhibited insulin-stimulated DNA and glycogen synthesis (
      • Kasus-Jacobi A.
      • Perdereau D.
      • Auzan C.
      • Clauser E.
      • Van Obberghen E.
      • Mauvais-Jarvis F.
      • Girard J.
      • Burnol A.F.
      ). Grb14 also inhibited IR substrate phosphorylation in vitro that was observed for Grb10 (
      • Bereziat V.
      • Kasus-Jacobi A.
      • Perdereau D.
      • Cariou B.
      • Girard J.
      • Burnol A.F.
      ). Mouse Grb10α overexpression in IGF-IR-transformed (but not in functional IGF-IR-deficient) mouse fibroblasts inhibited IGF-I-mediated cell growth, but not insulin-mediated cell growth in IR overexpressing cells (
      • Morrione A.
      • Valentinis B.
      • Resnicoff M.
      • Xu S.Q.
      • Baserga R.
      ). The authors' studies demonstrated a stimulatory mitogenic role of mouse Grb10δ in normal NIH 3T3 and baby hamster ovary fibroblasts in response to insulin, IGF-I, and platelet-derived growth factor by a number of complementary experimental strategies (
      • Wang J.
      • Dai H.
      • Yousaf N.
      • Moussaif M.
      • Deng Y.
      • Boufelliga A.
      • Rama Swamy O.
      • Leone M.E.
      • Riedel H.
      ). A stimulatory role was also supported by the observed inhibition of insulin and IGF-I-stimulated mitogenesis in response to microinjection of a putative dominant-negative Grb10γ peptide mimetic of the BPS and SH2 domains (
      • O'Neill T.J.
      • Rose D.W.
      • Pillay T.S.
      • Hotta K.
      • Olefsky J.M.
      • Gustafson T.A.
      ). A peptide mimetic of the Grb10 BPS domain inhibited substrate phosphorylation of activated IR and the catalytic activity of activated IGF-IR (
      • Stein E.G.
      • Gustafson T.A.
      • Hubbard S.R.
      ). In response to overexpression of human Grb10ζ in primary rat hepatocytes, some reduction of IR autophosphorylation, glycogen synthase activity, and glycogen synthesis was reported, whereas surprisingly, insulin-stimulated IRS-1 phosphorylation, PI 3-kinase activation, Akt/PKB activity, GSK3 activity, and ERK1/2 MAP kinase activity were reported unchanged (
      • Mounier C.
      • Lavoie L.
      • Dumas V.
      • Mohammas-Ali K.
      • Wu J.
      • Nantel A.
      • Bergeron J.J.M.
      • Thomas D.Y.
      • Posner B.I.
      ). Grb10 has been described as an important activator of Akt in particular in the signaling pathway downstream of the receptor tyrosine kinase c-Kit (
      • Jahn T.
      • Seipel P.
      • Urschel S.
      • Peschel C.
      • Duyster J.
      ). In contrast, partial interference of human Grb10ζ was reported with IRS-1, IRS-2, and Akt phosphorylation but not with IR autophosphorylation (
      • Wick K.R.
      • Werner E.D.
      • Langlais P.
      • Ramos F.J.
      • Dong L.Q.
      • Shoelson S.E.
      • Liu F.
      ). Experiments were based on overexpression of human Grb10ζ either in Chinese hamster ovary cells overexpressing IR or by adenovirus-mediated high level expression in mouse 3T3-L1 adipocytes but did not address the physiologic role of Grb10. Yeast-tri-hybrid studies implicated an interfering role of human Grb10ζ that required its SH2 domain in the association of IRS proteins with IR (
      • Wick K.R.
      • Werner E.D.
      • Langlais P.
      • Ramos F.J.
      • Dong L.Q.
      • Shoelson S.E.
      • Liu F.
      ). Using a lytic, recombinant adenovirus overexpression system, highly elevated levels of mouse Grb10δ in mouse 3T3-L1 adipocytes inhibited insulin-stimulated glucose uptake by 50%, whereas a mouse Grb10δ amino-terminal truncation mutant carrying only the BPS and SH2 domains did not affect glucose uptake under any conditions (
      • Mori K.
      • Giovannone B.
      • Smith R.J.
      ). Both Grb10 forms partially interfered with insulin-stimulated IRS-1/2, Akt, Shc, ERK1/2, and c-Cbl phosphorylation but not with IR phosphorylation (
      • Mori K.
      • Giovannone B.
      • Smith R.J.
      ). Key differences to the experiments we describe in this article lie in the 20-fold Grb10 overexpression levels and in the lytic adenoviral expression system that could possibly impact on cellular Grb10 function. It would be tempting to explain some of the reported experimental differences with the distinct employed Grb10 variants. Direct functional comparisons of Grb10 variants from different species have not yet been reported. However, in an initial comparison of human Grb10β human Grb10ζ and mouse Grb10δ in mouse L6 cells by cDNA expression, all variants comparably stimulated insulin-mediated metabolic responses including glycogen synthesis or glucose uptake.
      H. Xu and H. Riedel, unpublished results.
      It appears that the experimental host cell system (insulin-responsive versus fibroblast overexpressing IR), the employed mechanism of Grb10 expression (cell membrane-permeable peptides or cDNA expression versus lytic, high level recombinant adenovirus infection), and the measured endpoint (in vivo physiologic response versus in vitro molecular association or activation such as phosphorylation) have a major impact on the experimental outcome and interpretation of the respective result. The specific contribution of Grb10 appears highly dependent on the cellular context including the balance of other signaling mediators that define whether increased Grb10 levels will enhance or restrain a given response.
      Grb10 is subject to genomic imprinting with the majority of Grb10 expression arising from the maternally inherited allele in the mouse embryo, whereas the paternal allele is responsible for the majority of Grb10 expression in the adult mouse (
      • Hikichi T.
      • Kohda T.
      • Kaneko-Ishino T.
      • Ishino F.
      ). The recently reported disruption of the maternal Grb10 allele causes overgrowth of embryo and placenta, resulting in mice of a 30% increased birth weight (
      • Charalambous M.
      • Smith F.M.
      • Bennett W.R.
      • Crew T.E.
      • Mackenzie F.
      • Ward A.
      ). The gene disruption demonstrates in the mouse that Grb10 will constrain embryonal growth in a way that is released in the absence of functional Grb10. This study did not yet shed light on the role of Grb10 in metabolic insulin action except for a high glycogen content that was observed in liver hepatocytes and remains to be interpreted (
      • Charalambous M.
      • Smith F.M.
      • Bennett W.R.
      • Crew T.E.
      • Mackenzie F.
      • Ward A.
      ). In this context, Grb10 action was found essentially independent of insulin-like growth factor-2, indicating that imprinting acts on at least two major fetal growth axes and suggesting against a role of IGF-IR in Grb10 growth regulation (
      • Charalambous M.
      • Smith F.M.
      • Bennett W.R.
      • Crew T.E.
      • Mackenzie F.
      • Ward A.
      ). This interpretation is not supported by the reported interaction of Grb10 with ubiquitin ligase Nedd4 and the resulting ubiquitination and subsequent degradation of IGF-IR (
      • Vecchione A.
      • Marchese A.
      • Henry P.
      • Rotin D.
      • Morrione A.
      ). Two Grb10 partners have been reported that could participate in that process (
      • Giovannone B.
      • Lee E.
      • Laviola L.
      • Giorgino F.
      • Cleveland K.A.
      • Smith R.J.
      ). Grb10 gene disruption may potentially up-regulate other members of the superfamily such as Grb7 and Grb14 that may stimulate cell proliferation consistent with some of their reported cellular roles (reviewed in Refs.
      • Shen T.
      • Guan J.
      ,
      • Cariou B.
      • Bereziat V.
      • Moncoq K.
      • Kasus-Jacobi A.
      • Perdereau D.
      • Le Marcis V.
      • Burnol A.F.
      , and
      • Riedel H.
      ). The disruption of the Grb10 gene may also shift the balance to Grb7 or Grb14 that may usually compete for shared cellular targets with Grb10. The molecular mechanisms that cause overgrowth in response to Grb10 gene disruption and their interpretation remain unclear until a more comprehensive analysis will be available that includes the contribution of the Grb7 and Grb14 superfamily members.
      In combination, the findings of this report introduce mouse Grb10 as a critical component of the metabolic IR signaling complex that provides an alternative functional link between IR and p85 PI 3-kinase. This link parallels the established link of IR and p85 via IRS proteins because a direct association between Grb10 and IRS proteins could not be demonstrated. The role of Grb10 in PI 3-kinase regulation appears critical because dominant-negative Grb10 domains, in particular peptide mimetics of the SH2 domain, eliminated the metabolic response to insulin in differentiated 3T3-L1 adipocytes. This was consistently observed at the level of glycogen synthesis, glucose and amino acid transport, and lipogenesis. Alternatively, the same metabolic responses were substantially elevated by increased levels of Grb10. In summary, Grb10 represents a critical component in the IR signaling complex in the activation of PI 3-kinase that may have been overlooked in earlier studies in part based on its ubiquitous presence. The detailed dissection of its interaction with p85 and the resulting regulation of PI 3-kinase activity should help elucidate the complexity of the IR signaling mechanism. Future studies should address whether mouse Grb10 directly modulates additional downstream components in the metabolic insulin signal and whether it may affect the specificity of the insulin response. A direct functional comparison of the various Grb10 isoforms in an identical cellular and experimental background should help define common and distinct molecular roles.

      Acknowledgments

      We thank Minhchau Ha for expert assistance in the preparation of figures and Abdirahman Tahir Nur for general support. We are grateful to Dr. Nora Riedel for the critical discussion of experimental strategies and data.

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