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ERK Negatively Regulates the Epidermal Growth Factor-mediated Interaction of Gab1 and the Phosphatidylinositol 3-Kinase*

  • Cheng Fang Yu
    Correspondence
    To whom correspondence should be addressed: Dept. of Internal Medicine, Section of Nephrology, Yale University School of Medicine, 333 Cedar St., LMP 2093, New Haven, CT 06520-8062. Tel.: 203-785-7111; Fax: 203-785-7068
    Affiliations
    Department of Internal Medicine, Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut 06520
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  • Zhen-Xiang Liu
    Affiliations
    Department of Internal Medicine, Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut 06520
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  • Lloyd G. Cantley
    Affiliations
    Department of Internal Medicine, Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut 06520
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant DK54911 (to L. G. C.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:March 14, 2002DOI:https://doi.org/10.1074/jbc.M200732200
      We have examined the ability of epidermal growth factor (EGF)-stimulated ERK activation to regulate Grb2-associated binder-1 (Gab1)/phosphatidylinositol 3-kinase (PI3K) interactions. Inhibiting ERK activation with the MEK inhibitor U0126 increased the EGF-stimulated association of Gab1 with either full-length glutathione S-transferase-p85 or the p85 C-terminal Src homology 2 (SH2) domain, a result reproduced by co-immunoprecipitation of the native proteins from intact cells. This increased association of Gab1 and the PI3K correlates with an increase in PI3K activity and greater phosphorylation of Akt. This result is in direct contrast to what we have previously reported following HGF stimulation where MEK inhibition decreased the HGF-stimulated association of Gab1 and p85. In support of this divergent effect of ERK on Gab1/PI3K association following HGF and EGF stimulation, U0126 decreased the HGF-stimulated association of p85 and the Gab1 c-Met binding domain but did not alter the EGF-stimulated association of p85 and the c-Met binding domain. An examination of the mechanism of this effect revealed that the treatment of cells with EGF + U0126 increased the tyrosine phosphorylation of Gab1 as well as its association with another SH2-containing protein, SHP2. Furthermore, overexpression of a catalytically inactive form of SHP2 or pretreatment with pervanadate markedly increased EGF-stimulated Gab1 tyrosine phosphorylation. These experiments demonstrate that EGF and HGF-mediated ERK activation result in divergent effects on Gab1/PI3K signaling. HGF-stimulated ERK activation increases the Gab1/PI3K association, whereas EGF-stimulated ERK activation results in a decrease in the tyrosine phosphorylation of Gab1 and a decreased association with the PI3K. SHP2 is shown to associate with and dephosphorylate Gab1, suggesting that EGF-stimulated ERK might act through the regulation of SHP2.
      Grb2-associated binder-1 (Gab1)
      The abbreviations used are: Gab1
      Grb2-associated binder-1
      EGF
      epidermal growth factor
      EGFR
      EGF receptor
      PH
      pleckstrin homology
      PI3K
      phosphatidylinositol 3-kinase
      SH
      Src homology
      MAPK
      mitogen-activated protein kinase
      PI-3
      4,5-P3, phosphatidylinositol 3,4,5-trisphosphate
      HEK
      human embryonic kidney
      TBS-T
      Tris-buffered saline with Tween 20
      GST
      glutathione S-transferase
      ERK
      extracellular signal-regulated kinase
      MEK
      MAPK/extracellular signal-regulated kinase kinase
      SHP2-WT
      wild-type SHP2
      SHP-CS
      catalytically inactive SHP2
      HGF
      hepatocyte growth factor
      1The abbreviations used are: Gab1
      Grb2-associated binder-1
      EGF
      epidermal growth factor
      EGFR
      EGF receptor
      PH
      pleckstrin homology
      PI3K
      phosphatidylinositol 3-kinase
      SH
      Src homology
      MAPK
      mitogen-activated protein kinase
      PI-3
      4,5-P3, phosphatidylinositol 3,4,5-trisphosphate
      HEK
      human embryonic kidney
      TBS-T
      Tris-buffered saline with Tween 20
      GST
      glutathione S-transferase
      ERK
      extracellular signal-regulated kinase
      MEK
      MAPK/extracellular signal-regulated kinase kinase
      SHP2-WT
      wild-type SHP2
      SHP-CS
      catalytically inactive SHP2
      HGF
      hepatocyte growth factor
      has been identified in many cell types and appears to play a central role in multiple cell responses including proliferation, migration, tubulogenesis, cellular transformation, and apoptosis (
      • Weidner K.M., Di
      • Cesare S.
      • Sachs M.
      • Brinkmann V.
      • Behrens J.
      • Birchmeier W.
      ,
      • Holgado-Madruga M.
      • Emlet D.R.
      • Moscatello D.K.
      • Godwin A.K.
      • Wong A.J.
      ,
      • Kameda H.
      • Risinger J.I.
      • Han B.B.
      • Baek S.J.
      • Barrett J.C.
      • Abe T.
      • Takeuchi T.
      • Glasgow W.C.
      • Eling T.E.
      ,
      • Korhonen J.M.
      • Said F.A.
      • Wong A.J.
      • Kaplan D.R.
      ,
      • Maroun C.R.
      • Holgado-Madruga M.
      • Royal I.
      • Naujokas M.A.
      • Fournier T.M.
      • Wong A.J.
      • Park M.
      ,
      • Fixman E.D.
      • Holgado-Madruga M.
      • Nguyen L.
      • Kamikura D.M.
      • Fournier T.M.
      • Wong A.J.
      • Park M.
      ). Structural and functional studies suggest that Gab1 is a multisubstrate-docking protein functioning downstream of several receptor signaling pathways including the epidermal growth factor (EGF) receptor (EGFR), c-met, and the insulin receptor tyrosine kinases as well as cytokine receptors such as the gp130-associated interleukin-6 receptor and T and B cell antigen receptors (
      • Yu C.F.
      • Roshan B.
      • Liu Z.X.
      • Cantley L.G.
      ,
      • Cunnick J.M.
      • Mei L.
      • Doupnik C.A.
      • Wu J.
      ,
      • Takahashi-Tezuka M.
      • Yoshida Y.
      • Fukada T.
      • Ohtani T.
      • Yamanaka Y.
      • Nishida K.
      • Nakajima K.
      • Hibi M.
      • Hirano T.
      ,
      • Nishida K.
      • Yoshida Y.
      • Itoh M.
      • Fukada T.
      • Ohtani T.
      • Shirogane T.
      • Atsumi T.
      • Takahashi-Tezuka M.
      • Ishihara K.
      • Hibi M.
      • Hirano T.
      ). Similar to the Drosophila daughter of sevenless protein, DOS, and the insulin receptor substrate proteins 1 and 2 family, Gab1 consists of a PH domain at its N terminus, several proline-rich motifs in the C-terminus, and multiple tyrosine phosphorylation sites. But Gab1 is unique in that it contains a c-met binding domain (MBD) that includes the 13 amino acid c-met binding sequence, which mediates direct association with c-met (
      • Weidner K.M., Di
      • Cesare S.
      • Sachs M.
      • Brinkmann V.
      • Behrens J.
      • Birchmeier W.
      ,
      • Schaeper U.
      • Gehring N.H.
      • Fuchs K.P.
      • Sachs M.
      • Kempkes B.
      • Birchmeier W.
      ). The MBD also mediates indirect association with the activated EGFR via its proline-rich association with the SH3 domain of Grb2 (
      • Holgado-Madruga M.
      • Emlet D.R.
      • Moscatello D.K.
      • Godwin A.K.
      • Wong A.J.
      ,
      • Schaeper U.
      • Gehring N.H.
      • Fuchs K.P.
      • Sachs M.
      • Kempkes B.
      • Birchmeier W.
      ,
      • Roshan B.
      • Kjelsberg C.
      • Spokes K.
      • Eldred A.
      • Crovello C.S.
      • Cantley L.G.
      ). The PH domain has been found to be important for Gab1 localization and epithelial cellular morphogenesis in Madin-Darby canine kidney cells (
      • Maroun C.R.
      • Holgado-Madruga M.
      • Royal I.
      • Naujokas M.A.
      • Fournier T.M.
      • Wong A.J.
      • Park M.
      ), whereas neoplastic transformation in SHE cells has been found to correlate with the loss of this domain (
      • Kameda H.
      • Risinger J.I.
      • Han B.B.
      • Baek S.J.
      • Barrett J.C.
      • Abe T.
      • Takeuchi T.
      • Glasgow W.C.
      • Eling T.E.
      ).
      Following ligand binding, Gab1 associates with activated c-met or EGFR and is phosphorylated on specific tyrosine residues, in turn recruiting a series of SH2 domain-containing proteins that initiate intracellular signaling cascades. One of the most important signaling proteins found to associate with Gab1 in response to various stimuli is the phosphoinositide 3-kinase (PI3K). Studies have shown that the activation of the PI3K is involved in a wide range of cellular responses including cell proliferation, differentiation, and prevention of apoptosis (
      • Frevert E.U.
      • Bjorbaek C.
      • Venable C.L.
      • Keller S.R.
      • Kahn B.B.
      ,
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      ,
      • Leevers S.J.
      • Weinkove D.
      • MacDougall L.K.
      • Hafen E.
      • Waterfield M.D.
      ). We have previously demonstrated that the PI3K is required for HGF-mediated kidney epithelial cell migration and in vitro tubulogenesis (
      • Derman M.P.
      • Cunha M.J.
      • Barros E.J.
      • Nigam S.K.
      • Cantley L.G.
      ). The importance of Gab1 for this response has been demonstrated in work by Maroun et al. (
      • Maroun C.R.
      • Holgado-Madruga M.
      • Royal I.
      • Naujokas M.A.
      • Fournier T.M.
      • Wong A.J.
      • Park M.
      ) who found that the loss of association of Gab1 and the PI3K due to mutation of the PI3K binding sites in Gab1 results in a decrease of c-met-mediated tubulogenesis in Madin-Darby canine kidney cells. An association of the PI3K with Gab1 was also reported to be required for HGF-mediated cell survival and DNA repair (
      • Fan S.
      • Ma Y.X.
      • Gao M.
      • Yuan R.Q.
      • Meng Q.
      • Goldberg I.D.
      • Rosen E.M.
      ).
      Previously, it was believed that regulation of the PI3K association with Gab1 was mediated solely by receptor-dependent tyrosine phosphorylation of Gab1 on the PI3K SH2 binding motifs447YVPM451 and472YVPM476 in the MBD domain, and/or589YVPM at the carboxyl terminus. However, we have demonstrated that in addition to tyrosine phosphorylation, Gab1 is also phosphorylated on serine and threonine in response to ERK2 activation by HGF (
      • Roshan B.
      • Kjelsberg C.
      • Spokes K.
      • Eldred A.
      • Crovello C.S.
      • Cantley L.G.
      ). In vitro phosphorylation studies revealed that ERK2 primarily phosphorylates Gab1 in the MBD domain, and an examination of the Gab1 sequence for potential ERK binding and phosphorylation sites revealed that the476TP478 motif immediately following the472YVPM476 PI3K binding site was a high probability ERK1/2 phosphorylation site within the MBD domain of Gab1. In a more recent study, we found that the phosphorylation of both Tyr472 and Thr476 resulted in a higher affinity of a YVPMTP-containing peptide for the PI3K than did the phosphorylation of Tyr472 alone. Thus, ERK1/2-mediated phosphorylation of this site can initiate a novel regulation of the Gab1/PI3K interaction. This was confirmed by demonstrating that HGF-stimulated association of the PI3K with Gab1 was partially dependent on ERK activation (
      • Yu C.F.
      • Roshan B.
      • Liu Z.X.
      • Cantley L.G.
      ).
      Our recent determination that HGF-stimulated epithelial cell morphogenesis requires ERK1/2 activation provides a potential physiologic role for ERK-regulated PI3K activation (
      • Karihaloo A.
      • O'Rourke D.A.
      • Nickel C.
      • Spokes K.
      • Cantley L.G.
      ). Interestingly, in this same study, we found that EGF, but not HGF, activates ERK5 in addition to ERK1/2. The expression of a kinase-dead form of ERK5 in epithelial cells prevented EGF-stimulated morphogenesis, demonstrating that EGF and HGF use different MAPK signaling pathways for their morphogenic responses. Because the branching morphogenesis observed following EGF and HGF stimulation is phenotypically distinct, we decided to investigate the effects of EGF-stimulated ERK activation on the association of Gab1 with the PI3K. In contrast to the positive regulation of the Gab1/PI3K interaction that we found following HGF-stimulated ERK activation, EGF-stimulated ERK activation down-regulates the interaction of Gab1 and the PI3K. The investigation of the mechanism of this effect revealed that EGF-stimulated tyrosine phosphorylation of Gab1 was diminished in the setting of ERK activation, thereby decreasing the association of SH2-docking proteins.

      ACKNOWLEDGEMENTS

      We thank Lucia Rameh for the GST-p85 fusion protein constructs and Anton Bennett and Maria Kontaridis for the SHP2 constructs.

      REFERENCES

        • Weidner K.M., Di
        • Cesare S.
        • Sachs M.
        • Brinkmann V.
        • Behrens J.
        • Birchmeier W.
        Nature. 1996; 384: 173-176
        • Holgado-Madruga M.
        • Emlet D.R.
        • Moscatello D.K.
        • Godwin A.K.
        • Wong A.J.
        Nature. 1996; 379: 560-564
        • Kameda H.
        • Risinger J.I.
        • Han B.B.
        • Baek S.J.
        • Barrett J.C.
        • Abe T.
        • Takeuchi T.
        • Glasgow W.C.
        • Eling T.E.
        Mol. Cell. Biol. 2001; 21: 6895-6905
        • Korhonen J.M.
        • Said F.A.
        • Wong A.J.
        • Kaplan D.R.
        J. Biol. Chem. 1999; 274: 37307-37314
        • Maroun C.R.
        • Holgado-Madruga M.
        • Royal I.
        • Naujokas M.A.
        • Fournier T.M.
        • Wong A.J.
        • Park M.
        Mol. Cell. Biol. 1999; 19: 1784-1799
        • Fixman E.D.
        • Holgado-Madruga M.
        • Nguyen L.
        • Kamikura D.M.
        • Fournier T.M.
        • Wong A.J.
        • Park M.
        J. Biol. Chem. 1997; 272: 20167-20172
        • Yu C.F.
        • Roshan B.
        • Liu Z.X.
        • Cantley L.G.
        J. Biol. Chem. 2001; 276: 32552-32558
        • Cunnick J.M.
        • Mei L.
        • Doupnik C.A.
        • Wu J.
        J. Biol. Chem. 2001; 276: 24380-24387
        • Takahashi-Tezuka M.
        • Yoshida Y.
        • Fukada T.
        • Ohtani T.
        • Yamanaka Y.
        • Nishida K.
        • Nakajima K.
        • Hibi M.
        • Hirano T.
        Mol. Cell. Biol. 1998; 18: 4109-4117
        • Nishida K.
        • Yoshida Y.
        • Itoh M.
        • Fukada T.
        • Ohtani T.
        • Shirogane T.
        • Atsumi T.
        • Takahashi-Tezuka M.
        • Ishihara K.
        • Hibi M.
        • Hirano T.
        Blood. 1999; 93: 1809-1816
        • Schaeper U.
        • Gehring N.H.
        • Fuchs K.P.
        • Sachs M.
        • Kempkes B.
        • Birchmeier W.
        J. Cell Biol. 2000; 149: 1419-1432
        • Roshan B.
        • Kjelsberg C.
        • Spokes K.
        • Eldred A.
        • Crovello C.S.
        • Cantley L.G.
        J. Biol. Chem. 1999; 274: 36362-36368
        • Frevert E.U.
        • Bjorbaek C.
        • Venable C.L.
        • Keller S.R.
        • Kahn B.B.
        J. Biol. Chem. 1998; 273: 25480-25487
        • Kimura K.
        • Hattori S.
        • Kabuyama Y.
        • Shizawa Y.
        • Takayanagi J.
        • Nakamura S.
        • Toki S.
        • Matsuda Y.
        • Onodera K.
        • Fukui Y.
        J. Biol. Chem. 1994; 269: 18961-18967
        • Leevers S.J.
        • Weinkove D.
        • MacDougall L.K.
        • Hafen E.
        • Waterfield M.D.
        EMBO J. 1996; 15: 6584-6594
        • Derman M.P.
        • Cunha M.J.
        • Barros E.J.
        • Nigam S.K.
        • Cantley L.G.
        Am. J. Physiol. 1995; 268: F1211-F1217
        • Fan S.
        • Ma Y.X.
        • Gao M.
        • Yuan R.Q.
        • Meng Q.
        • Goldberg I.D.
        • Rosen E.M.
        Mol. Cell. Biol. 2001; 21: 4968-4984
        • Karihaloo A.
        • O'Rourke D.A.
        • Nickel C.
        • Spokes K.
        • Cantley L.G.
        J. Biol. Chem. 2001; 276: 9166-9173
        • Rauchman M.
        • Nigam S.
        • Delpire E.
        • Gullans S.
        Am. J. Physiol. 1993; 265: F416-F424
        • Davies S.P.
        • Reddy H.
        • Caivano M.
        • Cohen P.
        Biochem. J. 2000; 351: 95-105
        • Yart A.
        • Laffargue M.
        • Mayeux P.
        • Chretien S.
        • Peres C.
        • Tonks N.
        • Roche S.
        • Payrastre B.
        • Chap H.
        • Raynal P.
        J. Biol. Chem. 2001; 276: 8856-8864
        • Cunnick J.M.
        • Dorsey J.F.
        • Munoz-Antonia T.
        • Mei L.
        • Wu J.
        J. Biol. Chem. 2000; 275: 13842-13848
        • Alessi D.R.
        • Andjelkovic M.
        • Caudwell B.
        • Cron P.
        • Morrice N.
        • Cohen P.
        • Hemmings B.A.
        EMBO J. 1996; 15: 6541-6551
        • Barros E.J.
        • Santos O.F.
        • Matsumoto K.
        • Nakamura T.
        • Nigam S.K.
        Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4412-4416