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Epidermal Growth Factor-induced Phosphatidylinositol 3-Kinase Activation and DNA Synthesis

IDENTIFICATION OF Grb2-ASSOCIATED BINDER 2 AS THE MAJOR MEDIATOR IN RAT HEPATOCYTES*
  • Mei Kong
    Affiliations
    Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada
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  • Catherine Mounier
    Affiliations
    Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada
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  • Jiong Wu
    Affiliations
    Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada
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  • Barry I. Posner
    Correspondence
    To whom correspondence should be addressed: Polypeptide Hormone Laboratory, Strathocona Anatomy Building, 3640 University Street, Montreal, Quebec H3A 2B2, Canada. Tel.: 514-398-4101; Fax: 514-398-3923
    Affiliations
    Polypeptide Hormone Laboratory, Faculty of Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada
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  • Author Footnotes
    * This work was supported by the Medical Research Council and the National Cancer Institute of Canada as well as the Lady Davis Institute for Research at the Sir Mortimer B. Davis Jewish General Hospital, Montreal, Quebec. This work was also supported by the Cleghorn Fund at McGill University and the Maurice Pollack Foundation of Montreal.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:November 17, 2000DOI:https://doi.org/10.1074/jbc.M005621200
      In previous work we showed that the phosphatidylinositol 3-kinase (PI3-kinase), not the mitogen-activated protein kinase, pathway is necessary and sufficient to account for insulin- and epidermal growth factor (EGF)-induced DNA synthesis in rat hepatocytes. Here, using a dominant-negative p85, we confirmed the key role of EGF-induced PI3-kinase activation and sought to identify the mechanism by which this is effected. Our results show that EGF activates PI3-kinase with a time course similar to that of the association of p85 with three principal phosphotyrosine proteins (i.e. PY180, PY105, and PY52). We demonstrated that each formed a distinct p85-associated complex. PY180 and PY52 each constituted about 10% of EGF-activated PI3-kinase, whereas PY105 was responsible for 80%. PY105 associated with Grb2 and SHP-2, and although it behaved like Gab1, none of the latter was detected in rat liver. We therefore cloned a cDNA from rat liver, which was found to be 95% homologous to the mouse Grb2-associated binder 2 (Gab2) cDNA sequence. Using a specific Gab2 antibody, we demonstrated its expression in and association with p85, SHP-2, and Grb2 upon EGF treatment of rat hepatocytes. Gab2 accounted for most if not all of the PY105 species, since immunoprecipitation of Gab2 with specific antibodies demonstrated parallel immunodepletion of Gab2 and PY105 from the residual supernatants. We also found that the PI3-kinase activity associated with Gab2 was totally abolished by dominant negative p85. Thus, Gab2 appears to be the principal EGF-induced PY protein recruiting and activating PI3-kinase and mitogenesis.
      PI3-kinase
      phosphatidylinositol 3-kinase
      EGF
      epidermal growth factor
      EGFR
      EGF receptor
      Gab1 and Gab2
      Grb2-associated binder 1 and 2, respectively
      Grb2
      growth factor receptor bound 2
      SHP-2
      Src homology 2 domain-containing protein-tyrosine phosphatase-2
      SH2 and SH3
      Src homology 2 and 3 domain, respectively
      PCR
      polymerase chain reaction
      PAGE
      polyacrylamide gel electrophoresis
      IRS
      insulin receptor substrate
      IP
      immunoprecipitation
      WB
      Western blot
      Phosphatidylinositol 3-kinase (PI3-kinase)1 is an enzyme that phosphorylates the D-3 position of the inositol ring of PI to produce three novel phosphoinositides: phosphatidylinositol 3-monophosphate, phosphatidylinositol 3,4-biphosphate, and phosphatidylinositol 3,4,5-trisphosphate (
      • Auger K.R.
      • Serunian L.A.
      • Soltoff S.P.
      • Libby P.
      • Cantley L.C.
      ,
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.
      ). This enzyme is a heterodimer of a 110-kDa (p110) catalytic subunit and an 85-kDa (p85) regulatory subunit (
      • Carpenter C.L.
      • Duckworth B.C.
      • Auger K.R.
      • Cohen B.
      • Schaffhausen B.S.
      • Cantley L.C.
      ). p85 has two SH2 domains that bind to tyrosine-phosphorylated sites and an SH3 domain that binds proline-rich sequences on receptors or docking proteins (
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ,
      • Booker G.W.
      • Breeze A.L.
      • Downing A.K.
      • Panayotou G.
      • Gout I.
      • Waterfield M.D.
      • Campbell I.D.
      ). It has been demonstrated that p110 requires the binding of p85 to achieve full activation (
      • Holt K.H.
      • Olson L.
      • Moye-Rowley W.S.
      • Pessin J.E.
      ).
      Several studies, in various cell lines, suggest that the PI3-kinase pathway is important for both insulin (
      • Jhun B.H.
      • Rose D.W.
      • Seely B.L.
      • Rameh L.
      • Cantley L.
      • Saltiel A.R.
      • Olefsky J.M.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ) and epidermal growth factor (EGF)-induced mitogenesis (
      • Derossi D.
      • Williams E.J.
      • Green P.J.
      • Dunican D.J.
      • Doherty P.
      ,
      • Roche S.
      • Koegl M.
      • Courtneidge S.A.
      ). In primary rat hepatocytes, we previously demonstrated that the PI3-kinase, and not mitogen-activated protein kinase, pathway is necessary and sufficient to account for both insulin- and EGF-induced DNA synthesis (
      • Band C.J.
      • Mounier C.
      • Posner B.I.
      ).
      Several reports show that insulin achieves activation of PI3-kinase through the recruitment of p85 to tyrosine-phosphorylated IRS-1 and IRS-2 (
      • Backer J.M.
      • Myers Jr M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      ,
      • Hadari Y.R.
      • Tzahar E.
      • Nadiv O.
      • Rothenberg P.
      • Roberts C.T.
      • LeRoith Jr., D.
      • Yarden Y.
      • Zick Y.
      ,
      • Tobe K.
      • Tamemoto H.
      • Yamauchi T.
      • Aizawa S.
      • Yazaki Y.
      • Kadowaki T.
      ). In the case of EGF, studies in different cell lines have identified several possible mechanisms leading to PI3-kinase activation. Thus, in mouse fibroblast cell line overexpressing human EGFR (NRHER5), immunoprecipitates of EGFR were shown to contain PI3-kinase activity (
      • Thompson D.M.
      • Cochet C.
      • Chambaz E.M.
      • Gill G.N.
      ,
      • Bjorge J.D.
      • Chan T.O.
      • Antczak M.
      • Kung H.J.
      • Fujita D.J.
      ). In A431 cells and MDA-MB-468 breast cancer cell lines, tyrosine-phosphorylated ErbB3, a member of the EGFR family, was implicated in the activation of PI3-kinase upon EGF stimulation (
      • Kim H.H.
      • Sieke S.I.
      • Koland J.G.
      ,
      • Soltoff S.P.
      • Carraway III, K.L.
      • Prigent S.A.
      • Gullick W.G.
      • Cantley L.C.
      ). In PC12 and A549 cells, p120 cbl was found to associate with both SH2 and SH3 domain of p85, leading to activation of PI3-kinase, upon EGF stimulation (
      • Soltoff S.P.
      • Cantley L.C.
      ). Other studies have demonstrated that, in A431 cells, the recently cloned docking protein Grb2-associated binder 1 (Gab1) interacts with the p85 subunit of PI3-kinase following EGF (
      • Holgado-Madruga M.
      • Emlet D.R.
      • Moscatello D.K.
      • Godwin A.K.
      • Wong A.J.
      ). Another member of the Gab family, Gab2, was found to associate with p85 after treatment of hemopoietic cells with erythropoietin (
      • Wickrema A.
      • Uddin S.
      • Sharma A.
      • Chen F.
      • Alsayed Y.
      • Ahmad S.
      • Sawyer S.T.
      • Krystal G.
      • Yi T.
      • Nishada K.
      • Hibi M.
      • Hirano T.
      • Platanias L.C.
      ) and interleukin-3 (
      • Zhao C., Yu, D.H.
      • Shen R.
      • Feng G.S.
      ,
      • Nishida K.
      • Yoshida Y.
      • Itoh M.
      • Fukada T.
      • Ohtani T.
      • Shirogane T.
      • Atsumi T.
      • Takahashi-Tezuka M.
      • Ishihara K.
      • Hibi M.
      • Hirano T.
      ).
      The aim of this work was to clarify the mechanism of PI3-kinase activation following EGF stimulation of primary rat hepatocytes, a relevant physiological system. By overexpressing a dominant-negative p85, we confirmed the key role of PI3-kinase in EGF-induced DNA-synthesis. We next characterized three different p85-associated complexes generated by EGF treatment and identified Gab2 as a key molecule responsible for EGF-induced PI3-kinase activation in rat hepatocytes.

      DISCUSSION

      Using pharmacologic inhibitors of PI3-kinase and mitogen-activated protein kinase activation, we and others have demonstrated that the former and not latter pathway is both necessary and sufficient for insulin- and EGF-induced DNA synthesis (
      • Band C.J.
      • Mounier C.
      • Posner B.I.
      ,
      • Wymann M.P.
      • Bulgarelli-Leva G.
      • Zvelebil M.J.
      • Pirola L.
      • Vanhaesebroeck B.
      • Waterfield M.D.
      • Panayotou G.
      ,
      • Vlahos C.J
      • Matter W.F.
      • Hui K.Y.
      • Brown R.F.
      ,
      • Dudley D.T.
      • Pang L.
      • Decker S.J.
      • Bridges A.J.
      • Saltiel A.R.
      ). However, inhibitors may not always be sufficiently specific to warrant precise conclusions. Hence, wortmannin has also been shown to inhibit phosphatidylinositol 4-kinase as well as PI3-kinase (
      • Meyers R.
      • Cantley L.C.
      ). Using a dominant negative p85, in which the p110 binding site was deleted, we showed that both insulin- and EGF-induced DNA synthesis are totally abrogated (Fig. 1), confirming the essential role of PI3-kinase in mediating this effect in rat hepatocytes.
      Two general mechanisms for the recruitment and activation of PI3-kinase by growth factor receptors have been described in different transformed cell lines. The first involves the direct binding of the p85 regulatory subunit of PI3-kinase to PY receptor tyrosine kinases such as the platelet-derived growth factor (
      • Fantl W.J.
      • Escobedo J.A.
      • Martin G.A.
      • Turck C.W.
      • del Rosario M.
      • McCormick F.
      • Williams L.T.
      ), colony-stimulating factor-1 (
      • Kanagasundaram V.
      • Jaworowski A.
      • Hamilton J.A.
      ) and c-Met receptors (
      • Lee C.C.
      • Yamada K.M.
      ). The second mechanism comprises the recruitment to and activation of p85 by substrates of receptor tyrosine kinases (i.e. PY docking proteins) such as the IRS (
      • Backer J.M.
      • Myers Jr M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      ,
      • Hadari Y.R.
      • Tzahar E.
      • Nadiv O.
      • Rothenberg P.
      • Roberts C.T.
      • LeRoith Jr., D.
      • Yarden Y.
      • Zick Y.
      ,
      • Tobe K.
      • Tamemoto H.
      • Yamauchi T.
      • Aizawa S.
      • Yazaki Y.
      • Kadowaki T.
      ) and Gab protein families (
      • Holgado-Madruga M.
      • Emlet D.R.
      • Moscatello D.K.
      • Godwin A.K.
      • Wong A.J.
      ). The mechanism by which PI3-kinase becomes activated upon insulin stimulation has been well characterized (
      • Backer J.M.
      • Myers Jr M.G.
      • Shoelson S.E.
      • Chin D.J.
      • Sun X.J.
      • Miralpeix M.
      • Hu P.
      • Margolis B.
      • Skolnik E.Y.
      • Schlessinger J.
      ,
      • Hadari Y.R.
      • Tzahar E.
      • Nadiv O.
      • Rothenberg P.
      • Roberts C.T.
      • LeRoith Jr., D.
      • Yarden Y.
      • Zick Y.
      ,
      • Tobe K.
      • Tamemoto H.
      • Yamauchi T.
      • Aizawa S.
      • Yazaki Y.
      • Kadowaki T.
      ); however, the events consequent to EGF stimulation have been less clearly defined. As noted above, EGF activates PI3-kinase through recruitment to a range of PY proteins in a cell line-specific manner. In this paper, we demonstrate that PY proteins, especially that migrating at 105 kDa, play a key role in EGF-induced PI3-kinase activity in rat hepatocytes, a physiologically relevant system. Further analysis demonstrated that, upon EGF treatment, three distinct p85-associated complexes were formed, with the one containing the PY 105-kDa protein (identified as Gab2; Fig. 6) accounting for a large proportion of the activated PI3-kinase generated by EGF stimulation (Fig. 5).
      This study is the first to demonstrate that in rat hepatocytes EGF stimulates the formation of a complex composed of SHP-2, p105, p85, and Grb2, which accounts for over 80% of total EGF-induced PI3-kinase activity (Fig. 5).
      Based on our immunodepletion studies (Fig. 6), we conclude that PY105 is largely if not completely accounted for by Gab2. Gab2, a 100-kDa protein, has recently been cloned from mouse and human tissues (
      • Zhao C., Yu, D.H.
      • Shen R.
      • Feng G.S.
      ,
      • Nishida K.
      • Yoshida Y.
      • Itoh M.
      • Fukada T.
      • Ohtani T.
      • Shirogane T.
      • Atsumi T.
      • Takahashi-Tezuka M.
      • Ishihara K.
      • Hibi M.
      • Hirano T.
      ). It shows high homology with Gab1, a previously identified docking protein involved in growth factor signaling (
      • Korhonen J.M.
      • Said F.A.
      • Wong A.J.
      • Kaplan D.R.
      ,
      • Laffargue M.
      • Raynal P.
      • Yart A.
      • Peres C.
      • Wetzker R.
      • Roche S.
      • Payrastre B.
      • Chap H.
      ,
      • Maroun C.R.
      • Moscatello D.K.
      • Naujokas M.A.
      • Holgado-Madruga M.
      • Wong A.J.
      • Park M.
      ). Gab2 contains an N-terminal pleckstrin homology domain and proline-rich sequences as well as consensus PI 3-kinase, SHP-2, Grb2, and Crk tyrosine binding sites (
      • Nishida K.
      • Yoshida Y.
      • Itoh M.
      • Fukada T.
      • Ohtani T.
      • Shirogane T.
      • Atsumi T.
      • Takahashi-Tezuka M.
      • Ishihara K.
      • Hibi M.
      • Hirano T.
      ,
      • Gu H.
      • Pratt J.C.
      • Burakoff S.J.
      • Neel B.G.
      ). The direct association of Grb2 and p85, mediated by the SH3 domains of Grb2 and the proline-rich motifs of p85, has also been reported (
      • Wang J.
      • Auger K.R.
      • Jarvis L.
      • Shi Y.
      • Roberts T.M.
      ). However, the direct association of Grb2 and p85 in rat hepatocytes still needs to be established. Although SHP-2 was observed in anti-p85 immunoprecipitates in various cells, no direct association of SHP-2 and p85 has been reported. Rather, this association was probably mediated via a docking protein(s) (
      • Gesbert F.
      • Guenzi C.
      • Bertoglio J.
      ), such as Gab1 (
      • Ingham R.J.
      • Holgado-Madruga M.
      • Siu C.
      • Wong A.J.
      • Gold M.R.
      ) and Gab2 (
      • Gu H.
      • Pratt J.C.
      • Burakoff S.J.
      • Neel B.G.
      ). Indeed, on the basis of its similarity to Gab1 and homology with other docking molecules such as the IRSs (
      • Ando A.
      • Yonezawa K.
      • Gout I.
      • Nakata T.
      • Ueda H.
      • Hara K.
      • Kitamura Y
      • Noda Y.
      • Takenawa T.
      • Hirokawa N.
      ), it is likely that Gab2 works as a platform for the signaling complex consisting of p85, SHP-2, and Grb2. Our data also showed that dominant negative p85 fully inhibited both Grb2 and Gab2-associated PI3-kinase as well as the EGF-induced DNA synthesis. Taken together, these results argue for a critical role for the Gab2 protein in EGF-induced PI3-kinase activation and DNA synthesis in primary rat hepatocytes.
      Several reports have shown that, in hematopoietic cells, different stimuli (viz. interleukin-2, interleukin-3, and M-colony-stimulating factor) promote the association of PY proteins (95–110 kDa) with SHP-2, p85, and Grb2 (
      • Takahashi Y.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Gesbert F.
      • Guenzi C.
      • Bertoglio J.
      ,
      • Zhang S.
      • Broxmeyer H.E.
      ,
      • Gadina M.
      • Sudarshan C.
      • O'Shea J.J.
      ,
      • Carlberg K.
      • Rohrschneider L.R.
      ,
      • Craddock B.L.
      • Welham M.J.
      ). These reports are consistent with the possibility that the 95–110-kDa protein(s) is Gab2, which associates with p85, Grb2, and SHP-2 to play an important role in cytokine- and growth factor-regulated cell proliferation and differentiation.
      The function of ErbB3/p85-containing complex is unclear. Analysis of PI3-kinase activity in ErbB3 immunoprecipitates shows that it is less than 10% of the total EGF-induced PI3-kinase activity present in the Tyr(P) immunoprecipitates (data not shown). Since the association of p85 to ErbB3 does not lead to activation of a significant proportion of the PI3-kinase pool, we suggest that its main role might be to recruit p85 to the plasma membrane, in which compartment it may play a selective role. PY 52- and 46-kDa isoforms of Shc also associate with p85 in response to EGF in primary rat hepatocytes. As with ErbB3, this p85/Shc association, measured in Shc immunoprecipitates, represents less than 10% of the total PI3-kinase activation effected by EGF (data not shown). It also remains to be determined whether this association is direct or is via other proteins. However, we cannot see the association of ErbB3 with Shc, which suggests that p85 formed a complex with Shc distinct from that formed with ErbB3.
      In summary, we have demonstrated the critical role played by the recruitment of p85 to PY proteins in mediating EGF-induced PI3-kinase activity. We have identified three distinct p85-associated complexes that form in primary rat hepatocytes in response to EGF (Fig. 7). One complex contained ErbB3 and p85; the second contained p85 and Shc; and the third contained p85, Gab2, SHP-2, and Grb2. The last complex accounted for most EGF-induced PI3-kinase activation in rat hepatocytes. These findings point to a key role for Gab2 in effecting EGF-dependent biological functions such as mitogenesis.

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