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

Open AccessPublished:August 01, 2001DOI:https://doi.org/10.1074/jbc.M104493200
      Based on our previous observations that active ERK associates with and phosphorylates Gab1 in response to HGF, and the prediction that the ERK phosphorylation site is adjacent to one of the phosphatidylinositol 3-kinase (PI3K) SH2 binding motifs, we examined the possibility that ERK phosphorylation can regulate the Gab1/PI3K association. The HGF-mediated association of Gab1 with either full-length GST-p85 or its isolated N- or C-terminal SH2 domains was inhibited by ∼50% in the setting of ERK inhibition, a result confirmed by co-immunoprecipitation of the native proteins. A 14-amino acid peptide encoding472YVPMTP477 (one of the major p85 binding sites in Gab1 and the predicted ERK phosphorylation site) was synthesized with either phosphotyrosine alone (pY), or phosphotyrosine + phosphothreonine (pYT). In both pull-down assays and competition assays, pYT demonstrated a higher affinity for p85 than did pY alone. Finally, examination of the phosphorylation state of Akt after HGF stimulation revealed that ERK inhibition resulted in a decrease in Akt activation at both 5 and 10 min. These results suggest that activated ERK can phosphorylate Gab1 in response to HGF stimulation and thereby potentiate the Gab1/PI3K association and subsequent PI3K activation.
      HGF
      hepatocyte growth factor
      EGF
      epidermal growth factor
      PI3K
      phosphatidylinositol 3-kinase
      PI3
      4,5P3, phosphoinositide 3,4,5-trisphosphate
      PKC
      protein kinase C
      MBD
      Met binding domain
      ERK
      extracellular signal-regulated kinase
      MEK
      mitogen-activated protein kinase/ERK kinase
      GST
      glutathione S-transferase
      PAGE
      polyacrylamide gel electrophoresis
      PDGF
      platelet-derived growth factor
      PDGFR
      PDGF receptor
      PKB/Akt
      protein kinase B
      The docking protein Gab1 associates with several receptor tyrosine kinases known to induce cell morphogenesis, including the hepatocyte growth factor (HGF)1receptor, c-Met, and the epidermal growth factor (EGF) receptor. Following receptor activation, Gab1 associates and is tyrosine-phosphorylated, in turn recruiting multiple SH2 domain containing signal transducing proteins such as SHC, SHP2, phosphoinositide 3-kinase (PI3K), Crk/CRKL, and phospholipase Cγ (
      • Holgado-Madruga M.
      • Emlet D.R.
      • Moscatello D.K.
      • Godwin A.K.
      • Wong A.J.
      ,
      • Weidner K.M.
      • Di Cesare S.
      • Sachs M.
      • Brinkmann V.
      • Behrens J.
      • Birchmeier W.
      ,
      • Nguyen L.
      • Holgado-Madruga M.
      • Maroun C.
      • Fixman E.D.
      • Kamikura D.
      • Fournier T.
      • Charest A.
      • Tremblay M.L.
      • Wong A.J.
      • Park M.
      ,
      • Janez A.
      • Worrall D.S.
      • Imamura T.
      • Sharma P.M.
      • Olefsky J.M.
      ,
      • Cunnick J.M.
      • Dorsey J.F.
      • Munoz-Antonia T.
      • Mei L.
      • Wu J.
      ,
      • Ingham R.J.
      • Holgado-Madruga M.
      • Siu C.
      • Wong A.J.
      • Gold M.R.
      ). These proteins form an intricate signaling complex that regulates cell migration, morphology, and tubule formation. The critical nature of Gab1 signaling in normal cell biology has been demonstrated by the finding that Gab1-deficient mice die in utero (
      • Itoh M.
      • Yoshida Y.
      • Nishida K.
      • Narimatsu M.
      • Hibi M.
      • Hirano T.
      ) and display developmental defects in myotomes, placenta, and liver that recapitulate the loss of HGF and c-Met (
      • Sachs M.
      • Brohmann H.
      • Zechner D.
      • Muller T.
      • Hulsken J.
      • Walther I.
      • Schaeper U.
      • Birchmeier C.
      • Birchmeier W.
      ).
      The phenotypic effects of Gab1 signaling have been found to be critically dependent on Gab1 interactions with the PI3K. The PI3K is a heterodimer composed of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110). Two SH2 domains are located in the C-terminal region of the p85 subunit and have been shown to bind to membrane-associated tyrosine-phosphorylated proteins, resulting in recruitment of the PI3K to the membrane and activation of the lipid kinase activity of the 110-kDa subunit. This activity results in the formation of phosphoinositide 3,4,5-trisphosphate (PI3,4,5P3) at the membrane, which in turn serves to recruit and help activate such proteins as protein kinase C (PKC) and Akt (
      • Toker A.
      • Meyer M.
      • Reddy K.K.
      • Falck J.R.
      • Aneja R.
      • Aneja S.
      • Parra A.
      • Burns D.J.
      • Ballas L.M.
      • Cantley L.C.
      ,
      • Downward J.
      ). The activated PI3K has been demonstrated to result in the regulation of various cellular activities including proliferation (
      • Leevers S.J.
      • Weinkove D.
      • MacDougall L.K.
      • Hafen E.
      • Waterfield M.D.
      ), differentiation (
      • Kimura K.
      • Hattori S.
      • Kabuyama Y.
      • Shizawa Y.
      • Takayanagi J.
      • Nakamura S.
      • Toki S.
      • Matsuda Y.
      • Onodera K.
      • Fukui Y.
      ), and prevention of apoptosis (
      • Frevert E.U.
      • Bjorbaek C.
      • Venable C.L.
      • Keller S.R.
      • Kahn B.B.
      ). We have found that PI3K activity is critical for c-Met-mediated cell migration and in vitro tubulogenesis (
      • Derman M.P.
      • Cunha M.J.
      • Barros E.J.
      • Nigam S.K.
      • Cantley L.G.
      ), at least in part by activating PKC (
      • Derman M.
      • Toker A.
      • Hartwig J.
      • Spokes K.
      • Falck J.
      • Chen C.-S.
      • Cantley L.
      ). Another role for PI3K in morphogenic signaling has been demonstrated by the observation that the PH domain of Gab1 binds PI3,4,5P3, which serves to recruit Gab1 to the membrane following PI3K activation (
      • Holgado-Madruga M.
      • Emlet D.R.
      • Moscatello D.K.
      • Godwin A.K.
      • Wong A.J.
      ,
      • Maroun C.R.
      • Holgado-Madruga M.
      • Royal I.
      • Naujokas M.A.
      • Fournier T.M.
      • Wong A.J.
      • Park M.
      ,
      • Rodrigues G.A.
      • Falasca M.
      • Zhang Z.
      • Ong S.H.
      • Schlessinger J.
      ). Loss of the PH domain of Gab1 results in loss of epithelial morphogenesis following HGF stimulation, despite recruitment of Gab1 to the c-Met receptor and its phosphorylation (
      • Maroun C.R.
      • Holgado-Madruga M.
      • Royal I.
      • Naujokas M.A.
      • Fournier T.M.
      • Wong A.J.
      • Park M.
      ).
      We have recently demonstrated that in addition to SH2 and SH3 domain containing proteins, Gab1 also interacts with phosphorylated ERK1 and 2 following HGF or EGF stimulation of epithelial cells (
      • Roshan B.
      • Kjelsberg C.
      • Spokes K.
      • Eldred A.
      • Crovello C.S.
      • Cantley L.G.
      ). The association of ERK with Gab1 results in the phosphorylation of Gab1 on serine and threonine residues, primarily in the Met binding domain (MBD). This domain also includes one of three major consensus sequences for PI3K binding (472YVPM475), as well as a predicted ERK phosphorylation site immediately adjacent (472YVPMTP477). We therefore hypothesized that ERK phosphorylation of this site in vivomight result in secondary regulation of p85 SH2 domain binding to Gab1. In the present study, we confirm that HGF stimulation results in Gab1-p85 association, and demonstrate that this interaction is partly dependent on ERK activation. Furthermore, we show that inhibition of ERK activation not only decreases the association of p85 with Gab1 but also decreases the downstream phosphorylation of Akt.

      DISCUSSION

      In the present study we demonstrate that the addition of HGF to cells expressing the c-Met receptor results in both tyrosine phosphorylation of Gab1 as well as a decrease in the gel mobility of Gab1. The appearance of a slower mobility on SDS gels is typical of proteins that undergo phosphorylation on serine or threonine residues, particularly in the motif SP or TP. This is apparently because phosphorylation at these sites causes a change in the tertiary structure of the protein by creating a binding site for peptidyl-prolylcis/trans isomerases such as Pin1 (
      • Schutkowski M.
      • Bernhardt A.
      • Zhou X.Z.
      • Shen M.
      • Reimer U.
      • Rahfeld J.U.
      • Lu K.P.
      • Fischer G.
      ,
      • Lu K.P.
      • Hanes S.D.
      • Hunter T.
      ). In our previous study, we had found that the serine/threonine kinase ERK interacted with the MBD of Gab1 directly and could phosphorylate Gab lin vitro and in vivo resulting in an indistinguishable reduction in gel mobility (
      • Roshan B.
      • Kjelsberg C.
      • Spokes K.
      • Eldred A.
      • Crovello C.S.
      • Cantley L.G.
      ). Consistent with this data, we now show that inhibition of ERK activation prevents the HGF-mediated decrease in Gab1 gel mobility, arguing that ERK may directly phosphorylate Gab1 following HGF stimulation.
      ERK typically phosphorylates proteins at the motif PX(S/T)P (
      • Kennelly P.J.
      • Krebs E.G.
      ,
      • Davis R.J.
      ), often followed closely by an ERK binding motif consisting of FXFP (
      • Jacobs D.
      • Glossip D.
      • Xing H.
      • Muslin A.J.
      • Kornfeld K.
      ). Examining the sequence of Gab1 using the ScanSite program reveals one site in the Gab1 sequence that closely fits this preferred motif,472YVPMTPGTFDFS484. This site is in the Met binding domain (MBD) of Gab1, an 82-amino acid region of the protein that mediates the interaction of c-Met and Gab1 (
      • Weidner K.M.
      • Di Cesare S.
      • Sachs M.
      • Brinkmann V.
      • Behrens J.
      • Birchmeier W.
      ), which we have shown to selectively mediate ERK association and to be phosphorylated preferentially by ERK in vitro (
      • Roshan B.
      • Kjelsberg C.
      • Spokes K.
      • Eldred A.
      • Crovello C.S.
      • Cantley L.G.
      ). Interestingly, this ERK phosphorylation site is embedded in the classic PI3K SH2 association site, YVPM, and mutation of the tyrosine residue in this site has been shown to markedly decrease PI3K binding (
      • Schaeper U.
      • Gehring N.H.
      • Fuchs K.P.
      • Sachs M.
      • Kempkes B.
      • Birchmeier W.
      ,
      • Rocchi S.
      • Tartare-Deckert S.
      • Murdaca J.
      • Holgado-Madruga M.
      • Wong A.J.
      • Van Obberghen E.
      ). This suggests that ERK phosphorylation of this site might regulate the interaction of Gab1 and the PI3K.
      To test the role of endogenous ERK in the Gab1-PI3K association, we examined the ability of the p85 subunit of the PI3K to associate with Gab1 following HGF stimulation in the presence or absence of ERK inhibition. As expected, stimulation with HGF resulted in a marked increase in the affinity of p85 for Gab1, both in GST-p85 pull-down experiments and in co-immunoprecipitation of the native proteins. This increased affinity of Gab1 for p85 was reproduced equally for both the N- and C-terminal SH2 domains of p85, demonstrating its requirement for tyrosine phosphorylation of the Gab1 protein. However, pretreatment with the MEK inhibitor U0126 resulted in a substantial decrease in the p85-Gab1 interaction in both the pull-down and co-immunoprecipitation experiments, suggesting that ERK phosphorylation of Gab1 increases the affinity of Gab1 for p85. This result was unexpected, because ERK-mediated serine phosphorylation of the structurally similar docking protein, IRS-1, was found to have an opposite effect (
      • De Fea K.
      • Roth R.A.
      ).
      In the case of IRS-1, phosphorylation by several serine/threonine kinases, including ERK, PKC, and Akt, has been shown to decrease the affinity of IRS-1 for p85, at least in some cases by decreasing tyrosine phosphorylation of IRS-1 (
      • De Fea K.
      • Roth R.A.
      ,
      • De Fea K.
      • Roth R.A.
      ,
      • Li J.
      • DeFea K.
      • Roth R.A.
      ). Because the predicted ERK phosphorylation site in Gab1 is in the Met binding domain, we examined the possibility that inhibition of ERK activation was altering the association of c-Met and Gab1 and subsequently the tyrosine phosphorylation state of Gab1. We did not detect a difference in the association between Gab1 and its c-Met adapter protein Grb2, nor did we find a change in the total tyrosine phosphorylation state of Gab1 following HGF stimulation in the presence or absence of MEK inhibition. Thus it does not appear that treatment with U0126 prevents HGF-mediated c-Met activation or c-Met-dependent tyrosine phosphorylation of Gab1. Of note, because Gab1 is known to be potentially phosphorylated on at least 8 tyrosine residues (
      • Lehr S.
      • Kotzka J.
      • Herkner A.
      • Klein E.
      • Siethoff C.
      • Knebel B.
      • Noelle V.
      • Bruning J.C.
      • Klein H.W.
      • Meyer H.E.
      • Krone W.
      • Muller-Wieland D.
      ), a selective loss of tyrosine phosphorylation of one of the residues critical for p85 association might not be detected by this method.
      To determine whether phosphorylation of threonine 476 (the predicted ERK phosphorylation site in Gab1) alters the affinity of tyrosine 472 (the most important residue for p85 binding to Gab1 (
      • Rocchi S.
      • Tartare-Deckert S.
      • Murdaca J.
      • Holgado-Madruga M.
      • Wong A.J.
      • Van Obberghen E.
      )) for binding the SH2 domain of p85, three peptides based on the 14 amino acids encompassing this site were synthesized. As expected, the non-phosphorylated peptide failed to interact with p85, whereas pY472 pulled-down p85 at concentrations as low as 1 pm. However, in both simple pull-down experiments and competition experiments using the native p85 biding site in the PDGFR, the dual-phosphorylated peptide (pYT) displayed a higher affinity for p85 than did pY. Thus, dual phosphorylation on both tyrosine and threonine at the predicted ERK phosphorylation site in Gab1 results in a higher affinity for p85 binding than does tyrosine phosphorylation alone. The antibody used in these experiments can recognize both p85α and β, and the association of these isoforms with Gab1 is not well distinguished using the gel separation techniques presented. Therefore the association of either p85α, p85β, or both may be up-regulated in this setting. Conversely, the dual phosphorylation of Gab1 could result in a decreased affinity of Gab1 for an independent PI3K regulatory isoform such as p55.
      To test whether this proposed dual phosphorylation of Gab1 actually results in an alteration in PI3K downstream signaling, we examined the effects of inhibition of ERK activation on Akt phosphorylation. Akt is a PH domain containing serine/threonine kinase that is recruited to the membrane by the PI3K product PI3,4,5P3 where it is phosphorylated and activated by the membrane-associated kinase PDK1, resulting in enhanced cell survival (
      • Alessi D.R.
      • Andjelkovic M.
      • Caudwell B.
      • Cron P.
      • Morrice N.
      • Cohen P.
      • Hemmings B.A.
      ). Using an antibody specific for the activated state of Akt (anti-pT308), we found that activated Akt was detectable within 2 min following HGF stimulation. In the absence of U0126 treatment, the phosphorylation state of Akt increased further at 5 and 10 min, consistent with a further increase in PI3,4,5P3 production at the membrane. In the setting of ERK inhibition, the initial increase in phospho-Akt was not altered, but the subsequent further increase at 5 and 10 min was entirely abolished.
      Taken together, these data are most consistent with the model that c-Met activation results in Gab1 recruitment to the receptor followed by phosphorylation of Gab1 at multiple tyrosine residues, including472YVPM475. This results in association of PI3K with Gab1 and local production of PI3,4,5P3. In concert, HGF induces ERK activation with the subsequent association of Gab1 and ERK resulting in phosphorylation of Gab1 at Thr476in the PI3K docking site. This dually phosphorylated motif then can act as a higher affinity site for the p85 subunit of the PI3K, leading to a further recruitment and/or stabilization of PI3K at the membrane and subsequently greater activation of PI3,4,5P3-dependent signaling pathways. The increase in Akt phosphorylation observed in the absence of ERK inhibition suggests yet another pathway whereby ERK activation may help promote cell survival and prevent apoptosis.
      In conclusion, the HGF-mediated interaction of Gab1 and PI3K is dependent on tyrosine phosphorylation by the c-Met receptor, but is further enhanced by ERK-mediated phosphorylation of Gab1, possibly at Thr476. Threonine phosphorylation at this site results in a higher affinity of the PI3K binding site472YVPMTP477 for p85, whereas inhibition of ERK activation decreased the p85 association with Gab1 and decreased the activation of the downstream kinase Akt. Because activation of the PI3K is critical for epithelial cell migration and morphogenesis, this result may in part explain our prior observations that HGF-mediated epithelial cell migration and tubule formation are dependent on MAPK activation (
      • Karihaloo A.
      • O'Rourke D.A.
      • Nickel C.
      • Spokes K.
      • Cantley L.G.
      ).

      Acknowledgments

      We thank Lewis Cantley, Lucia Rameh, and Saskia Brackmann for their help and advice.

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