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Insulin Receptor Substrate (IRS)-1 and IRS-2 Are Tyrosine-phosphorylated and Associated with Phosphatidylinositol 3-Kinase in Response to Brain-derived Neurotrophic Factor in Cultured Cerebral Cortical Neurons*

  • Masashi Yamada
    Correspondence
    Research Fellow of the Japan Society for the Promotion of Science. To whom correspondence and reprint requests should be addressed: Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan. Tel.: 81-6-879-8625; Fax: 81-6-879-8626
    Affiliations
    Institute for Protein Research, Osaka University, 3–2 Yamadaoka, Suita, Osaka 565, Japan
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  • Hiroshi Ohnishi
    Affiliations
    Mitsubishi Kasei Institute of Life Science, 11 Minamiooya, Machida, Tokyo 194, Japan
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  • Shin-ichiro Sano
    Affiliations
    Mitsubishi Kasei Institute of Life Science, 11 Minamiooya, Machida, Tokyo 194, Japan
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  • Atsushi Nakatani
    Affiliations
    Institute for Protein Research, Osaka University, 3–2 Yamadaoka, Suita, Osaka 565, Japan
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  • Toshihiko Ikeuchi
    Affiliations
    Institute for Protein Research, Osaka University, 3–2 Yamadaoka, Suita, Osaka 565, Japan
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  • Hiroshi Hatanaka
    Affiliations
    Institute for Protein Research, Osaka University, 3–2 Yamadaoka, Suita, Osaka 565, Japan
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  • Author Footnotes
    * This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan.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 28, 1997DOI:https://doi.org/10.1074/jbc.272.48.30334
      Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, promotes differentiation and survival of various types of neurons in the central nervous system. BDNF binds to and activates the tyrosine kinase receptor, TrkB, initiating intracellular signaling and exerting its effects. Phosphatidylinositol 3-kinase (PI3-K), which has been implicated in promotion of neuronal survival by neurotrophic factors, is a component in the signaling pathway of BDNF. We examined how BDNF activates PI3-K in cultured cerebral cortical neurons. We found that insulin receptor substrate (IRS)-1 and -2 are involved in the BDNF signaling pathway that activates PI3-K. IRS-1 and -2 were tyrosine-phosphorylated and bound to PI3-K in response to BDNF. This BDNF-stimulated signaling via IRS-1 and -2 was inhibited by K-252a, an inhibitor of Trk tyrosine kinase. In addition, signaling via IRS-1 and -2 was markedly sustained as well as the BDNF-induced tyrosine phosphorylation of TrkB. On the other hand, we observed no association of PI3-K with TrkB in response to BDNF. These results indicate that the activation of TrkB by BDNF induces the activation of PI3-K via IRS-1 and -2 rather than by a direct interaction of TrkB with PI3-K in cultured cortical neurons.
      Neurotrophins, including nerve growth factor, brain-derived neurotrophic factor (BDNF),
      The abbreviations used are: BDNF, brain-derived neurotrophic factor; NT, neurotrophin; PI3-K, phosphatidylinositol 3-kinase; IRS, insulin receptor substrate; IGF, insulin-like growth factor; BSA, bovine serum albumin; NGF, nerve growth factor; MAP, mitogen-activated protein; SH2, Src homology 2.
      1The abbreviations used are: BDNF, brain-derived neurotrophic factor; NT, neurotrophin; PI3-K, phosphatidylinositol 3-kinase; IRS, insulin receptor substrate; IGF, insulin-like growth factor; BSA, bovine serum albumin; NGF, nerve growth factor; MAP, mitogen-activated protein; SH2, Src homology 2.
      neurotrophin (NT)-3, and NT-4/5, are important for regulation of differentiation, survival, and plasticity of various types of neurons (
      • Abiru Y.
      • Nishio C.
      • Hatanaka H.
      ,
      • Akaneya Y.
      • Tsumoto T.
      • Hatanaka H.
      ,
      • Kubo T.
      • Nonomura T.
      • Enokido Y.
      • Hatanaka H.
      ,
      • Nonomura T.
      • Nishio C.
      • Lindsay R.M.
      • Hatanaka H.
      ,
      • Nonomura T.
      • Kubo T.
      • Oka T.
      • Shimoke K.
      • Yamada M.
      • Enokido Y.
      • Hatanaka H.
      ,
      • Takei N.
      • Sasaoka K.
      • Inoue K.
      • Takahashi M.
      • Endo Y.
      • Hatanaka H.
      ,
      • Takei N.
      • Sasaoka K.
      • Higuchi H.
      • Endo Y.
      • Hatanaka H.
      ,
      • Lewin G.R.
      • Barde Y.A.
      ,
      • Thoenen H.
      ). Neurotrophins bind to and activate the Trk family of receptor tyrosine kinases to exert their effects (
      • Barbacid M.
      ,
      • Bothwell M.
      ). NGF primarily binds to TrkA, BDNF and NT-4/5 bind to TrkB, and NT-3 binds to TrkC. Trks form homodimers following ligand binding, resulting in autophosphorylation on tyrosine residues, which is required for both catalytic and signaling activities (
      • Greene L.A.
      • Kaplan D.R.
      ,
      • Kaplan D.R.
      • Stephens R.M.
      ,
      • Lemmon M.A.
      • Schlessinger J.
      ,
      • Segal R.A.
      • Greenberg M.E.
      ,
      • van der Geer P.
      • Hunter T.
      • Lindberg R.A.
      ).
      Phosphatidylinositol 3-kinase (PI3-K) is activated by the neurotrophins as well as by various growth factors, including epidermal growth factor, platelet-derived growth factor, and hepatocyte growth factor (
      • Cochet C.
      • Filhol O.
      • Payrastre B.
      • Hunter T.
      • Gill G.N.
      ,
      • Graziani A.
      • Gramaglia D.
      • Cantley L.C.
      • Comoglio P.M.
      ,
      • Raffioni S.
      • Bradshaw R.A.
      ). In addition, several studies using PC12 cells and cultured cerebellar granule neurons have indicated that the activation of PI3-K is important for the survival-promoting effects of neurotrophic factors (
      • Yao R.
      • Cooper G.M.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Shimoke K.
      • Kubo T.
      • Numakawa T.
      • Abiru Y.
      • Enokido Y.
      • Takei N.
      • Ikeuchi T.
      • Hatanaka H.
      ). PI3-K is a heterodimer composed of an 85-kDa regulatory subunit (p85) and a 110-kDa catalytic subunit (p110) (
      • Escobedo J.A.
      • Navankasattusas S.
      • Kavanaugh W.M.
      • Milfay D.
      • Fried V.A.
      • Williams L.T.
      ,
      • Hiles I.D.
      • Otsu M.
      • Volinia S.
      • Fry M.J.
      • Gout I.
      • Dhand R.
      • Panayotou G.
      • Ruiz Larrea F.
      • Thompson A.
      • Totty N.F.
      • Hsuan J.J.
      • Courtheidge S.A.
      • Parker P.J.
      • Waterfield M.D.
      ,
      • Ruiz-Larrea F.
      • Vicendo P.
      • Yaish P.
      • End P.
      • Panayotou G.
      • Fry M.J.
      • Morgan S.J.
      • Thompson A.
      • Parker P.J.
      • Waterfield M.D.
      ). PI3-K is activated by association of p85 with phosphotyrosine residues on target proteins via SH2 domains of p85 (
      • Carpenter C.L.
      • Auger K.R.
      • Chanudhuri M.
      • Yoakim M.
      • Schaffhausen B.
      • Shoelson S.
      • Cantley L.C.
      ). PI3-K is activated by platelet-derived growth factor and hepatocyte growth factor through its binding to specific phosphotyrosine residues on autophosphorylated platelet-derived growth factor and hepatocyte growth factor receptors, respectively (
      • Cooper J.A.
      • Kashishian A.
      ,
      • Klippel A.
      • Escobedo J.A.
      • Fantl W.J.
      • Williams L.T.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Maina F.
      • Longati P.
      • Panayotou G.
      • Dhand R.
      • Waterfield M.D.
      • Comoglio P.M.
      ,
      • Ponzetto C.
      • Bardelli A.
      • Zhen Z.
      • Maina F.
      • dalla Zonca P.
      • Giordano S.
      • Graziani A.
      • Panayotou G.
      • Comoglio P.M.
      ). However, the mechanism by which neurotrophins activate PI3-K remains unclear. There have been contradictory reports regarding whether NGF activates PI3-K via a direct association of PI3-K with the autophosphorylated TrkA or via an indirect interaction of PI3-K with the activated TrkA (
      • Obermeier A.
      • Lammers R.
      • Wiesmuller K.H.
      • Jung G.
      • Schlessinger J.
      • Ullrich A.
      ,
      • Ohmichi M.
      • Decker S.J.
      • Saltiel A.R.
      ,
      • Soltoff S.P.
      • Rabin S.L.
      • Cantley L.C.
      • Kaplan D.R.
      ).
      Insulin receptor substrate (IRS)-1 is well known as a binding protein to PI3-K (
      • 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.
      • White M.F.
      ,
      • Keller S.R.
      • Lienhard G.E.
      ,
      • Waters S.B.
      • Pessin J.E.
      ). IRS-1 is a major substrate of the insulin receptor that possesses tyrosine kinase activity. PI3-K binds to specific phosphotyrosine residues on IRS-1, resulting in its activation. Recently, IRS-2 has been purified as a protein that associates with SH2 domains of PI3-K and has been shown to have sequence homology with IRS-1 (
      • Waters S.B.
      • Pessin J.E.
      ,
      • Patti M.-E.
      • Sun X.-J.
      • Bruening J.C.
      • Araki E.
      • Lipes M.A.
      • White M.F.
      • Kahn C.R.
      ,
      • Sun X.J.
      • Wang L.M.
      • Zhang Y.T.
      • Yenush L.
      • Myers Jr., M.G.
      • Glasheen E.
      • Lane W.S.
      • Pierce J.H.
      • White M.F.
      ). IRS-2 is also tyrosine-phosphorylated in response to insulin and binds to and activates PI3-K. IRS-1 and -2 are utilized in intracellular signaling pathways of insulin-like growth factor-1 (IGF-1); growth hormone; interleukin-2, -4, and -7; interferon α and γ, and leukemia inhibitory factor as well as in the insulin-activated pathway (
      • Argetsinger L.S.
      • Hsu G.W.
      • Myers Jr., M.G.
      • Billestrup N.
      • White M.F.
      • Carter-Su C.
      ,
      • Argetsinger L.S.
      • Norstedt G.
      • Billestrup N.
      • White M.F.
      • Carter-Su C.
      ,
      • Uddin S.
      • Yenush L.
      • Sun X.-J.
      • Sweet M.E.
      • White M.F.
      • Platanias L.C.
      ,
      • Johnston J.A.
      • Wang L.-M.
      • Hanson E.P.
      • Sun X.-J.
      • White M.F.
      • Oakes S.A.
      • Pierce J.H.
      • O'Shea J.J.
      ,
      • Platanias L.C.
      • Uddin S.
      • Yetter A.
      • Sun X.-J.
      • White M.F.
      ).
      Here, we found that BDNF stimulates tyrosine phosphorylation of IRS-1 and -2 in cultured cerebral cortical neurons. In addition, the tyrosine-phosphorylated IRS-1 and -2 are associated with the protein and activity of PI3-K. These results suggest that IRS-1 and -2 are components in the signaling pathway of BDNF and are involved in the activation of PI3-K in cultured cortical neurons.

      DISCUSSION

      Our results using cultured cerebral cortical neurons provided evidence for the presence of a novel signaling pathway of neurotrophins, i.e. IRS-1 and -2 are components of the neurotrophin-induced signaling pathway. IRS-1 and -2 are known to be phosphorylated on tyrosine residues by the receptors for insulin and IGF-1. No receptor tyrosine kinases except for the insulin and IGF-1 receptors have been reported to utilize IRS-1 or -2. The insulin receptor recognizes IRS-1 as a substrate through the interaction between the phosphotyrosine-binding domain on IRS-1 and the tyrosine-phosphorylated NPXY motif in the juxtamembrane domain of the insulin receptor (
      • Chen D.
      • Van Horn D.J.
      • White M.F.
      • Backer J.M.
      ,
      • O'Neill T.J.
      • Craparo A.
      • Gustafson T.A.
      ). IRS-2 also has a phosphotyrosine-binding domain, and it has been reported to be recognized by the insulin receptor in a similar way to IRS-1 (
      • Sawka-Verhelle D.
      • Tartare-Deckert S.
      • White M.F.
      • Van Obberghen E.
      ). Trks also have the NPXY motif in their juxtamembrane domain. In addition, Trks belong to the insulin receptor subfamily according to sequence homology of their tyrosine kinase domains (
      • Hanks S.K.
      • Quinn A.M.
      • Hunter T.
      ). Therefore, we consider that Trks as well as the insulin receptor directly phosphorylate IRS-1 and -2. On the other hand, we observed neither NGF- nor BDNF-induced tyrosine phosphorylation of IRS-1 and -2 in PC12 cells stably expressing TrkB (data not shown). The cultured cortical neurons expressed over 8-fold more TrkB protein than the TrkB-expressing PC12 cells (data not shown). However, the amounts of TrkA and TrkB proteins in the TrkB-expressing PC12 cells were thought to be almost the same as judged from the levels of their neurotrophin-induced tyrosine phosphorylation (data not shown). Therefore, the differences in amounts of Trk proteins between the cortical neurons and the PC12 cells may have caused the distinct responses in the neurotrophin-induced tyrosine phosphorylation of IRS-1 and -2. Alternatively, the neurotrophin-stimulated tyrosine phosphorylation of IRS-1 and -2 in cultured cortical neurons may require a signaling molecule that is not expressed in PC12 cells.
      The insulin-induced tyrosine-phosphorylated forms of IRS-1 and -2 bind to the protein-tyrosine phosphatase SHP-2 and the small adaptor protein GRB-2 as well as to PI3-K (
      • Waters S.B.
      • Pessin J.E.
      ,
      • Sugimoto S.
      • Wandless T.J.
      • Shoelson S.E.
      • Neel B.G.
      • Walsh C.T.
      ,
      • Ward C.W.
      • Gough K.H.
      • Rashke M.
      • Wan S.S.
      • Tribbick G.
      • Wang J.
      ). However, SHP-2 and GRB-2 were not coprecipitated by the anti-IRS-1 or -2 antibodies in response to neurotrophins in cultured cortical neurons (data not shown). The anti-IRS-1 antibody is raised against a synthetic peptide corresponding to a carboxyl-terminal amino acid sequence very close to the SHP-2-binding tyrosine residue on IRS-1, and the anti-IRS-2 antibody is raised against that including the SHP-2-binding tyrosine residue on IRS-2. The anti-IRS-1 and -2 antibodies might not precipitate the complex between SHP-2 and IRS-1 or -2 because of steric interference by SHP-2 in the association of IRS-1 or -2 with the antibodies or because of exclusion of SHP-2 binding to IRS-1 and -2 by the antibodies. On the other hand, we could not detect the 180-kDa tyrosine-phosphorylated protein, which was observed in the anti-PI3-K immunoprecipitates and was thought to be IRS-1 and/or -2, in the anti-SHP-2 immunoprecipitates in response to neurotrophins in cultured cortical neurons (data not shown). The Trk tyrosine kinases might have different specificity for tyrosine residues on IRS-1 and -2 from other tyrosine kinases including the insulin receptor, i.e. the tyrosine residues in the SHP-2 and GRB-2 binding sites may be phosphorylated by the insulin receptor but not by Trks, resulting in a lack of neurotrophin-induced formation of complexes between SHP-2 or GRB-2 and IRS-1 or -2. In contrast, the tyrosine residues in the PI3-K binding sites are thought to be phosphorylated by both Trks and the insulin receptor. In support of the different specificities among the tyrosine kinases, it has been reported that the insulin and IGF-1 receptors have varying specificities for individual tyrosine residues on IRS-1 in vitro (
      • Xu B.
      • Bird V.G.
      • Miller W.T.
      ). IRS-1 and -2 might show a variety of tyrosine-phosphorylated forms in response to different factors, utilizing different subsets of signaling molecules.
      Neurotrophic factors induce the sustained activation of the mitogen-activated protein (MAP) kinase pathway (
      • Marshall C.J.
      ,
      • Qui M.S.
      • Green S.H.
      ,
      • Traverse S.
      • Gomez N.
      • Paterson H.
      • Marshall C.
      • Cohen P.
      ). The sustained activation of MAP kinases is thought to be a feature of their neurotrophic action. We also observed that the BDNF-induced activation of MAP kinases in the cultured cortical neurons was sustained for at least 5 h after the addition of BDNF (data not shown). Here, we showed that the BDNF-stimulated signaling via IRS-1 and -2 was sustained in the cultured cortical neurons (Fig. 5). To exert neurotrophic effects, BDNF may not only induce the sustained activation of MAP kinases but also stimulate sustained signaling via IRS-1 and -2, which probably leads to sustained activation of PI3-K. Interestingly, the tyrosine phosphorylation of IRS-2 and its association with PI3-K increased again at 5 h after the addition of BDNF, although the phosphorylation and association of IRS-1 gradually declined after the increase at 5 min and did not rise again. The tyrosine phosphorylation of TrkB in response to BDNF also displayed a similar increase to that of IRS-1 (data not shown). Therefore, it is thought that the second increase in IRS-2 signaling was not due to the tyrosine kinase activity of TrkB. The second increase in the tyrosine phosphorylation of IRS-2 might require a tyrosine kinase other than TrkB, the activity of which shows a delayed increase. The expression of this tyrosine kinase and/or a regulatory protein of this kinase may be up-regulated by BDNF. Additionally, we observed that the amount of the IRS-2 protein with the higher molecular weight increased. Therefore, the biphasic response of IRS-2 might be due to the up-regulation of expression of the IRS-2 protein. In addition, IRS-2 displayed mobility shifts, which became larger after the peak of its tyrosine phosphorylation levels at 5 min. This delayed mobility shift of IRS-2 might have resulted from its phosphorylation on serine and/or threonine residues, but not on tyrosine residues, by a kinase that shows delayed activation in response to BDNF. Since the biphasic response of IRS-2 was reproducible, we suggest that some interesting regulatory mechanisms in the neurotrophin-induced signaling is implicated in this phenomenon.
      We observed that the anti-PI3-K antibody coprecipitated several tyrosine-phosphorylated proteins (180, 120, 95, and 70 kDa) in response to BDNF in cultured cerebral cortical neurons (Fig. 1). We assumed that these tyrosine-phosphorylated proteins bind to the SH2 domains of p85 regulatory subunit and activate PI3-K. From the present results, the 180-kDa protein is thought to be IRS-1 and/or -2. We could detect no tyrosine-phosphorylated proteins other than IRS-1 or -2 in the anti-IRS-1 or -2 immunoprecipitates. Therefore, the tyrosine-phosphorylated proteins with apparent molecular masses of 120, 95, and 70 kDa in the PI3-K precipitates might activate PI3-K independently of IRS-1 or -2. It is assumed that BDNF-induced signaling in the cultured cortical neurons has redundant pathways to activate PI3-K. In this study, we showed that the pathway via IRS-1 or -2 is one of the PI3-K-activating pathways in BDNF-induced signaling in cultured cortical neurons.

      Acknowledgments

      We thank Dr. R. Lindsay (Regeneron Pharmaceutical Co.) for the kind gifts of BDNF and NT-3; Dr. S. Koizumi (Novartis Pharma K. K.) for the generous gift of anti-TrkB monoclonal antibody; and Dr. M. Takahashi (Mitsubishi Kasei) for valuable advice and encouragement throughout this work.

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