Evidence for a Direct Interaction between Insulin Receptor Substrate-1 and Shc

doi:10.1074/jbc.272.27.17166

Evidence for a Direct Interaction between Insulin Receptor Substrate-1 and Shc*

From the Centre de Recherche sur l’Endocrinologie Moléculaire et le Développement du CNRS, UPR 1511, 92190 Meudon, France and the ^§INSERM U145, Avenue de Valombrose, 06107 Nice Cedex 2, France

Abstract

Insulin receptor substrate-1 (IRS-1) and Shc are two proteins implicated in intracellular signal transduction. They are activated by an increasing number of extracellular signals, mediated by receptor tyrosine kinases, cytokine receptors, and G protein-coupled receptors. In this study we demonstrate that Shc interacts directly with IRS-1, using the yeast two-hybrid system and an in vitrointeraction assay. Deletion analysis of the proteins to map the domains implicated in this interaction shows that the phosphotyrosine binding domain of Shc binds to the region of IRS-1 comprising amino acids 583–661. An in vitro association assay, performed with or without activation of tyrosine kinases, gives evidence that tyrosine phosphorylation of IRS-1 and Shc drastically improves the interaction. Site-directed mutagenesis on IRS-1 583–693 shows that the asparagine, but not the tyrosine residue of the N⁶²⁵GDY⁶²⁸motif domain, is implicated in the IRS-1-Shc-phosphotyrosine binding interaction. Mutation of another tyrosine residue, Tyr⁶⁰⁸, also induced a 40% decrease in the interaction. This study, describing a phosphotyrosine-dependent interaction between IRS-1 and Shc, suggests that this association might be important in signal transduction.

Footnotes

↵* This work was supported in part by grants from the Fondation de la Recherche Médicale and by Grant 1248 from the Association pour la Recherche sur le Cancer.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.
↵‡ Recipient of a student fellowship from the Ministère de la Recherche.
↵¶ To whom correspondence should be addressed: Centre de Recherche sur l’Endocrinologie Moléculaire et le Développement du CNRS, UPR 1511, 9 rue Jules Hetzel, 92190 Meudon, France. Tel.: 33-1-45-07-50-72; Fax: 33-1-45-07-58-90; E-mail:burnol{at}cnrs-bellevue.fr.
↵1 The abbreviations used are: IRS, insulin receptor substrate; SH2 domain, Src homology 2 domain; PTB domain, phosphotyrosine binding domain; IR, insulin receptor; GST, glutathioneS-transferase; Gal4 BD, Gal4 binding domain; Gal4 AD, Gal4 activation domain; PAGE, polyacrylamide gel electrophoresis; CH1, collagen homologous region; PIPES, 1,4-piperazinediethanesulfonic acid.
- Received April 23, 1997.