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Src-family Tyrosine Kinases in Activation of ERK-1 and p85/p110-phosphatidylinositol 3-Kinase by G/CCKBReceptors*

  • Laurence Daulhac
    Affiliations
    From the Groupe de Recherche de Biologie et Pathologie Digestives, INSERM U.151, CHU Rangueil, 1 avenue J. Poulhes, Institut Louis Bugnard, Batiment L3, 31403 Toulouse, France
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  • Aline Kowalski-Chauvel
    Affiliations
    From the Groupe de Recherche de Biologie et Pathologie Digestives, INSERM U.151, CHU Rangueil, 1 avenue J. Poulhes, Institut Louis Bugnard, Batiment L3, 31403 Toulouse, France
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  • Lucien Pradayrol
    Affiliations
    From the Groupe de Recherche de Biologie et Pathologie Digestives, INSERM U.151, CHU Rangueil, 1 avenue J. Poulhes, Institut Louis Bugnard, Batiment L3, 31403 Toulouse, France
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  • Nicole Vaysse
    Affiliations
    From the Groupe de Recherche de Biologie et Pathologie Digestives, INSERM U.151, CHU Rangueil, 1 avenue J. Poulhes, Institut Louis Bugnard, Batiment L3, 31403 Toulouse, France
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  • Catherine Seva
    Correspondence
    To whom correspondence should be addressed: INSERM U.151, Groupe de Recherche de Biologie et Pathologie Digestives, CHU Rangueil, 1 avenue J. Poulhes, Institut Louis Bugnard, Bat. L3, 31403 Toulouse, France. Tel.: 33 561322408; Fax: 33 561322403;
    Affiliations
    From the Groupe de Recherche de Biologie et Pathologie Digestives, INSERM U.151, CHU Rangueil, 1 avenue J. Poulhes, Institut Louis Bugnard, Batiment L3, 31403 Toulouse, France
    Search for articles by this author
  • Author Footnotes
    * This work was supported by funds from INSERM, Association pour la Recherche contre le Cancer Grant 9540, and the Conseil Régional Midi Pyrénées Grant 97001932.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:July 16, 1999DOI:https://doi.org/10.1074/jbc.274.29.20657
      We have analyzed in Chinese hamster ovary cells the upstream mediators by which the G protein-coupled receptor, gastrin/CCKB, activates the extracellular-regulated kinases (ERKs) and p85/p110-phosphatidylinositol 3-kinase (PI 3-kinase) pathways. Overexpression of an inhibitory mutant of Shc completely blocked gastrin-stimulated Shc·Grb2 complex formation but partially inhibited ERK-1 activation by this peptide. Expression of Csk, which inactivates Src-family kinases, totally inhibited gastrin-induced Src-like activity detected in anti-Src and anti-Shc precipitates but diminished by 50% Shc phosphorylation and ERK-1 activation. We observed a rapid tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src. This hypothesis was supported by the inhibition of gastrin-induced Src·IRS-1 complex formation and IRS-1 phosphorylation in Csk-transfected cells. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation was abolished by Csk. Our results demonstrate the existence of two mechanisms in gastrin-mediated ERKs activation. One requires Shc phosphorylation by Src-family kinases, and the other one is independent of these two proteins. They also indicate that tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin.
      Gastrin was initially characterized as the major hormonal regulator of gastric acid secretion. This polypeptide also functions as a growth factor on the gastrointestinal tract in vivo (
      • Johnson L.R.
      ). Its trophic effects have also been observed on several cancer cell lines derived from colon stomach and pancreas (
      • Kusyk C.J.
      • McNeil N.O.
      • Johnson L.R.
      ,
      • Seva C.
      • Scemama J.L.
      • Bastie M.J.
      • Pradayrol L.
      • Vaysse N.
      ,
      • Seva C.
      • Dickinson C.J.
      • Yamada T.
      ). Gastrin effects have been shown to be mediated by the gastrin/CCKB(G/CCKB)
      The abbreviations G/CCKB
      gastrin/CCKB
      ERK
      extracellular-regulated kinase
      GPCR
      G protein-coupled receptor
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PtdIns
      phosphatidylinositols
      IRS
      insulin receptor substrate
      CHO
      Chinese hamster ovary
      IP
      immunoprecipitation
      IB
      immunoblot
      1The abbreviations G/CCKB
      gastrin/CCKB
      ERK
      extracellular-regulated kinase
      GPCR
      G protein-coupled receptor
      PI 3-kinase
      phosphatidylinositol 3-kinase
      PtdIns
      phosphatidylinositols
      IRS
      insulin receptor substrate
      CHO
      Chinese hamster ovary
      IP
      immunoprecipitation
      IB
      immunoblot
      receptor, which belongs to the family of G protein-coupled receptors (
      • Wank S.A.
      • Pisegna J.R.
      • Weerth A.
      ,
      • Kopin A.S.
      • Lee Y.M.
      • Mc Bride E.W.
      • Miller L.J.
      • Kolakowski L.F.
      • Beinborn M.
      ).
      Extracellular-regulated kinase-1 and -2 (ERK-1 and ERK-2) are two members of the mitogen-activated protein kinase family known to play an important role in cell proliferation. They have been shown to be activated by a variety of receptors including receptor tyrosine kinases and G protein-coupled receptors (GPCRs). Their activation by growth factor receptors with intrinsic tyrosine kinase activity has been well documented. The best understood mechanism involves the recruitment of the Grb2·Sos or Shc·Grb2·Sos complexes to tyrosine-phosphorylated receptors. These complexes subsequently activate the following cascade: Ras/Raf/ERK kinases/ERKs (
      • Serger R.
      • Krebs E.G.
      ).
      Src-family tyrosine kinases have been proposed to serve as intermediates between GPCR and ERK activation. However, their involvement in this transduction pathway seems to be highly receptor- and cell type-specific. In some cells, lysophosphatidic acid and angiotensin receptors can activate ERKs via a mechanism that involves the tyrosine phosphorylation of Shc proteins by Src-family kinases (
      • Luttrell L.M.
      • Hawes B.E.
      • Van Biesen T.
      • Luttrell D.K.
      • Lansing T.J.
      • Lefkowitz R.J.
      ,
      • Dikic I.
      • Tokiwa G.
      • Lev S.
      • Courtneidge S.A.
      • Schlessinger J.
      ,
      • Daub H.
      • Wallasch C.
      • Lankenau A.
      • Herrlich A.
      • Ullrich A.
      ,
      • Zou Y.
      • Komuro I.
      • Yamazaki T.
      • Kudoh S.
      • Aikawa R.
      • Zhu W.
      • Shiojima I.
      • Hiroi Y.
      • Tobe K.
      • Kadowaki T.
      • Yazaki Y.
      ). In other cell types, neither Src nor Shc are required for ERK activation by these ligands (
      • Kranenburg O.
      • Verlaan I.
      • Hordijk P.L.
      • Moolenaar W.H.
      ). The mechanism leading to ERK activation by α1-B, α2-A adrenergic receptors, muscarinic acetylcholine, or bradykinin receptors also involves Src and Shc (
      • Dikic I.
      • Tokiwa G.
      • Lev S.
      • Courtneidge S.A.
      • Schlessinger J.
      ,
      • Della Rocca G.J.
      • Van Biesen T.
      • Daaka Y.
      • Luttrell D.K.
      • Luttrell L.M.
      • Lefkowitz R.J.
      ,
      • Wan Y.
      • Kurosaki T.
      • Huang X.-Y.
      ), whereas thrombin or formylmethionylleucylphenylalanine induce the ERK cascade independently of these two proteins (
      • Chen Y.H.
      • Grall D.
      • Salcini A.E.
      • Pelicci P.G.
      • Pouyssegur J.
      • Van Obberghen-Schilling E.
      ,
      • Torres M.
      • Ye R.D.
      ). We and others have recently shown that gastrin modulates ERK-1/2 activity; however, the contribution of Src-family kinases and Shc proteins has never been studied (
      • Taniguchi T.
      • Matsui T.
      • Ito M.
      • Murayama T.
      • Tsukamoto T.
      • Katakami Y.
      • Chiba T.
      • Chihara K.
      ,
      • Seva C.
      • Kowalski-Chauvel A.
      • Blanchet J.S.
      • Vaysse N.
      • Pradayrol L.
      ,
      • Seufferlein T.
      • Withers D.J.
      • Broad S.
      • Herget T.
      • Walsh J.H.
      • Rozengurt E.
      ).
      Phosphatidylinositol 3-kinases (PI 3-kinases) are lipid kinases that phosphorylate phosphatidylinositols (PtdIns) at the D-3 position of the inositol ring. Several PI 3-kinase isoforms have been described in the literature. The first cloned PI 3-kinase was a heterodimer composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit, which has been implicated in the regulation of mitogenesis and cell transformation (
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfray D.
      • Fried V.A.
      • Williams L.T.
      ,
      • Skolnick E.Y.
      • Margolis B.
      • Mohamnudi M.
      • Lowenstein E.
      • Fischer R.
      • Drepps A.
      • Ullrich A.
      • Schlessinger J.
      ,
      • Otsu M.
      • Hiles I.
      • Gout I.
      • Fry M.J.
      • Ruiz-Larrea F.
      • Panayoto G.
      • Thompson A.
      • Dhanal R.
      • Hsuan J.
      • Totty N.
      • Smith A.D.
      • Morgan S.J.
      • Courtneidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ). This enzyme has been found to be associated and activated by growth factor receptors (
      • Coughlin S.R.
      • Escobedo J.A.
      • Williams L.T.
      ,
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • Del Rosario M.
      • McCormick F.
      • Williams L.T.
      ). In addition, inhibition of PI 3-kinase activity by specific inhibitors or antibodies results in blockage of growth factor-induced cell proliferation (
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ,
      • Vemuri G.S.
      • Rittenhouse S.E.
      ). Activation of the p85/p110-PI 3-kinase by tyrosine kinase receptors such as platelet-derived growth factor or colony-stimulating factor-1 receptors involves the binding of the SH2 domain (for Src-Homology 2) of p85 to specific phosphotyrosine-containing sequences of the receptors (
      • Kashishian A.
      • Kazlaukas A.
      • Cooper J.A.
      ,
      • Reedijk M.
      • Liu X.
      • Van Der Geer P.
      • Letwin K.
      • Waterfield M.D.
      • Hunter T.
      • Pawson T.
      ). In contrast, the interaction of p85 with a tyrosine-phosphorylated intermediate protein, the insulin receptor substrate-1 (IRS-1), represents a mechanism for p85/p110-PI 3-kinase activation by insulin or insulin growth factor-1 (
      • White M.F.
      • Kahn C.R.
      ,
      • Giorgetti S.
      • Ballotti R.
      • Kowalski-Chauvel A.
      • Tartare S.
      • Van Obberghen E.
      ). Quite recently, novel forms of PI 3-kinases have been characterized. They do not interact with p85 and are directly activated by GPCRs and βγ subunits of G proteins (
      • Stephens L.
      • Smrcka A.
      • Cooke F.T.
      • Jackson T.R.
      • Sternweis P.C.
      • Hawkins P.T.
      ,
      • Thomason P.A.
      • James S.R.
      • Casey P.J.
      • Downes C.P.
      ,
      • Stoyanov B.
      • Volinia S.
      • Hanck T.
      • Rubio I.
      • Loubtchenkov M.
      • Malek D.
      • Stoyanova S.
      • Vanhaesebroeck B.
      • Dhand R.
      • Nurnberg B.
      ).
      Several laboratories including ours have reported that some GPCRs such as the G/CCKB are capable of activating the p85/p110-PI 3-kinase (
      • Hu Z.W.
      • Shi X.Y.
      • Lin R.Z.
      • Hoffman B.B.
      ,
      • Saward L.
      • Zahradka P.
      ,
      • Kowalski-Chauvel A.
      • Pradayrol L.
      • Vaysse N.
      • Seva C.
      ). However, very little is known about the mechanism of activation.
      In this study, we have analyzed the contribution of Src-family tyrosine kinases to the activation of ERK-1 or p85/p110-PI 3-kinase pathways by gastrin. We report here that Shc tyrosine phosphorylation by Src-family tyrosine kinases and Shc·Grb2 complex formation are early events in ERKs activation via the GPCR, G/CCKB. Similarly, Src-family tyrosine kinases, by phosphorylating IRS-1, play an essential role in the gastrin-mediated p85/p110-PI 3-kinase-signaling cascade.

      DISCUSSION

      The intracellular mechanisms activated by the G/CCKBreceptors are not completely elucidated; particularly the early events upstream of gastrin-activated signaling pathways remain to be characterized. The important role of Src-family tyrosine kinases in the transmission of mitogenic signals induced by growth factors whose receptors possess intrinsic tyrosine kinase activity is well documented (
      • Courtneidge S.A.
      • Fumagalli S.
      • Koegl M.
      • Super-Furga G.
      • Twamley-Stein G.M.
      ). In quiescent NIH-3T3 fibroblasts, kinase-inactive Src prevents platelet-derived growth factor-induced DNA synthesis. In a similar fashion, cells expressing inactive forms of Src and Fyn are unable to respond to proliferative signals. Overexpression of Src enhances the mitogenic response to epidermal growth factor treatment, whereas expression of Src inhibitory mutants decreases the response.
      In the present study, we demonstrate that gastrin, acting through a GPCR, rapidly and transiently stimulates the enzymatic activity of Src. We also analyzed the downstream targets phosphorylated and activated by this tyrosine kinase following gastrin stimulation. The increase in Src-like kinase activity observed in anti-Shc immunoprecipitates from gastrin-stimulated cells suggested that Shc may be a direct substrate of Src, phosphorylated on tyrosine residues in response to gastrin. This hypothesis was supported by the inhibition of gastrin-induced Shc phosphorylation and Shc·Grb2 complex formation in CHO cells transfected with Csk, known to inactivate Src-family tyrosine kinases. Our data also demonstrate that maximal activation of ERK-1 by gastrin requires Shc phosphorylation by Src-family tyrosine kinases. Indeed, we observed a decrease of gastrin-induced ERK-1 activation in CHO cells expressing Csk or a dominant inhibitory mutant of Shc. However, the partial inhibition obtained in these transfected cells suggests the existence of an additional pathway for gastrin-mediated ERK-1 stimulation independent of Shc and Src-family tyrosine kinases.
      The focal adhesion kinase-related protein tyrosine kinase (PYK2) as well as Bruton's tyrosine kinase, which belongs to a family of Pleckstrin homology domain-containing tyrosine kinases, have also been proposed to link GPCRs to the activation of the ERKs cascade (
      • Dikic I.
      • Tokiwa G.
      • Lev S.
      • Courtneidge S.A.
      • Schlessinger J.
      ,
      • Wan Y.
      • Bence K.
      • Hata A.
      • Kurosaki T.
      • Veillette A.
      • Huang X.Y.
      ). However, it seems that these nonreceptor tyrosine kinases interact with Src-family kinases to regulate their activity (
      • Dikic I.
      • Tokiwa G.
      • Lev S.
      • Courtneidge S.A.
      • Schlessinger J.
      ,
      • Cheng G.
      • Ye Z.S.
      • Baltimore D.
      ,
      • Mahajan S.
      • Fargnoli J.
      • Burkhardt A.L.
      • Kut S.A.
      • Saouaf S.J.
      • Bolen J.B.
      ). Therefore, they probably do not contribute to the Src-independent pathway, which relays the signal from G/CCKB receptors to the activation of ERK-1. Kranenburg et al. (
      • Kranenburg O.
      • Verlaan I.
      • Hordijk P.L.
      • Moolenaar W.H.
      ) also observe a regulation of the ERK pathway by GPCRs that do not involve Src and Shc proteins. Their findings suggest that a wortmannin-sensitive PI 3-kinase, whose activity is independent on Src-family kinases, links Gi-coupled receptors to ERKs activation. In the present study, we demonstrate in CHO cells transfected with the G/CCKB receptor that gastrin-mediated PI 3-kinase activation is totally dependent on Src-family tyrosine kinases. Consequently, PI 3-kinase cannot play a role in the Src-independent pathway that leads to ERK-1 activation by gastrin. Various isoforms of protein kinase C are capable of directly activating Raf-1 by phosphorylation (
      • Cacace A.M.
      • Ueffing M.
      • Philipp A.
      • Han E.K.
      • Kolch W.
      • Weinstein I.B.
      ,
      • Cai H.
      • Smola U.
      • Wixler V.
      • Eisenmann-Tappe I.
      • Diaz-Meco M.T.
      • Moscat J.
      • Rapp U.
      • Cooper G.M.
      ). Raf-1 subsequently phosphorylates the ERK kinases, which in turn activates the ERKs. This pathway, which requires neither the activation of tyrosine kinases nor the formation of the complex Shc·Grb2·Sos·Ras, may be involved in the Src-independent pathway that links G/CCKB receptors to ERK-1 activation.
      Recently, several studies have reported the activation of the p85/p110 isoform of PI 3-kinase by GPCRs including chemoattractant receptors, angiotensin receptors, and adrenergic receptors (
      • Hu Z.W.
      • Shi X.Y.
      • Lin R.Z.
      • Hoffman B.B.
      ,
      • Saward L.
      • Zahradka P.
      ,
      • Ptasznik A.
      • Prossnitz E.R.
      • Yoshikawa D.
      • Smrcka A.
      • Traynor-Kaplan A.E.
      • Bokoch G.M.
      ). However, very little is known about the molecular basis of this activation. Activation of the enzyme in chemoattractant-stimulated neutrophils seems to involve direct binding of the Src-related kinase, Lyn, to the p85 subunit of PI 3-kinase. This interaction might occur via the SH3 domain (for Src-Homology 3) of Lyn and the proline-rich domain of the p85 subunit as described previously for the activation of the PI 3-kinase by B cell antigen receptor (
      • Ptasznik A.
      • Prossnitz E.R.
      • Yoshikawa D.
      • Smrcka A.
      • Traynor-Kaplan A.E.
      • Bokoch G.M.
      ,
      • Ptasznik A.
      • Traynor-Kaplan A.E.
      • Bokoch G.M.
      ,
      • Pleiman C.M.
      • Hertz W.M.
      • Cambier J.C.
      ). In the present study, we describe a different mechanism for the activation of the p85/p110-PI 3-kinase by Src-family kinases, which uses IRS-1 as a docking molecule. Indeed, we demonstrate by Western blot that gastrin induces a rapid tyrosine phosphorylation of IRS-1 as well as the association of this protein with Src. We also observed an increase in Src-like kinase activity in anti-IRS-1 immunoprecipitates from gastrin-stimulated cells, suggesting that IRS-1 may be a direct substrate of Src, phosphorylated on tyrosine residues in response to gastrin. This hypothesis was supported by the inhibition of gastrin-induced Src·IRS-1 association and IRS-1 phosphorylation in CHO cells transfected with Csk. In addition, the increase in PI 3-kinase activity measured in anti-p85 or anti-IRS-1 precipitates following gastrin stimulation were abolished by Csk overexpression. The tyrosine phosphorylation of IRS-1 in response to thrombin, which binds to GPCRs, has also been observed (
      • Rao G.N.
      • Delafontaine P.
      • Runge M.S.
      ). However, the role of this phosphorylation remains to be determined. Angiotensin-1 receptors also belong to the family of GPCRs. They have been shown to mediate the tyrosine phosphorylation of IRS-1 via JAK2 tyrosine kinase, which associates with AT1 receptor and IRS-1 after angiotensin stimulation. Surprisingly, in this study an inhibition of the PI 3-kinase activity was observed after the association of the p85 regulatory subunit to IRS-1 (
      • Velloso L.A.
      • Folli F.
      • Sun X.J.
      • White M.F.
      • Saad M.J.A.
      • Kahn C.R.
      ). In contrast, our observations suggest that the tyrosine phosphorylation of IRS-1 by Src-family kinases could lead to the recruitment and the activation of the p85/p110-PI 3-kinase in response to gastrin.

      Acknowledgments

      We thank Drs. I. Baldari and P. G. Pelicci (Siena, Italy) for the pL-SH2-Shc-SN vector, Dr. S. Roche (Montpellier, France) for the cDNA encoding Csk, Drs. Y. Le Marchand-Brustel and J. F. Tanti (Nice, France) for antibodies to p85, and Drs. E. Van Obberghen and I. Mothe (Nice, France) for anti-IRS-1 antibodies.

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