Evidence for a direct interaction between insulin receptor substrate-1 and Shc

Insulin receptor substrate-1 (IRS-I) and She are two proteins implicated ill 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 She interacts directly with IRS-1, using the yeast two-hybrid system and an in. vitro interaction assay. Deletion analysis of the proteins to map the domains implicated in this interaction shows that the phosphotyrosine binding domain of She binds to the region of IRS-1 comprising amino acids 583-661. An irt vitro association assay, performed with or without activation of tyrosine kinases, gives evidence that tyrosine phosphorylation of IRS-1 and She drastically improves the interaction. Site-directed mutagenesis on IRS-I 583-693 shows that the asparagine, hut not the tyrosine residue of the N(625)GDY(628)motif domain, is implicated in the IRS-1-Shc-phosphotyrosine binding interaction. Mutation of another tyrosine residue, Tyr(608) also induced a 40% decrease in the interaction. This study, describing a phosphotyrosine-dependent interaction between IRS-1 and She, suggests that this association might be important in signal transduction.