Insulin stimulates association of insulin receptor substrate-1 with the protein abundant Src homology/growth factor receptor-bound protein 2.

Insulin activates the ras proto-oncogene product p21ras (Ras) by stimulating conversion of the inactive GDP-bound form of Ras to the active GTP-bound form. The protein ASH (for abundant Src homology) (Matuoka, K., Shibata, M., Yamakawa, A., and Takenawa, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9015-9019) is composed of one Src homology (SH)2 and two SH3 domains and highly homologous to the Caenorhabditis elegans protein sem-5 that couples a tyrosine kinase to a Ras protein. We have studied an interaction of ASH with insulin-stimulated tyrosine-phosphorylated proteins in Chinese hamster ovary cells overexpressing human insulin receptors (CHO-HIR cells). In an anti-ASH (alpha ASH) immunoprecipitates, we detected a 170-kDa phosphoprotein that was recognized by an anti-phosphotyrosine antibody and an anti-insulin receptor substrate 1 antibody (alpha IRS-1) from the insulin-stimulated [32P]orthophosphate-labeled CHO-HIR cells. We failed to detect the tyrosine phosphorylation of the protein ASH. These data suggested that insulin stimulates IRS-1.ASH complex formation in intact cells. Incubation of an ASH fusion protein with the lysates of insulin-stimulated CHO-HIR cells revealed that the fusion protein of ASH was able to bind the tyrosine-phosphorylated 170-kDa protein that was recognized by alpha IRS-1. We also demonstrated that fusion protein of ASH was able to bind the fusion protein of tyrosine-phosphorylated IRS-1 fragments, suggesting that ASH is able to bind tyrosine-phosphorylated IRS-1 directly. These data suggest that IRS-1.ASH complex formation may play a role in coupling the insulin receptor kinase to a Ras signaling pathway. Furthermore, we observed an insulin-stimulated phosphatidylinositol (PI) 3-kinase activity in alpha ASH immunoprecipitates, suggesting the formation of an ASH.IRS-1.PI 3-kinase complex. This complex formation was detected as early as 10 s after insulin stimulation in intact CHO-HIR cells. This is the first report that supports the notion that IRS-1 binds several signal transducing molecules containing SH2 domains, thus serves as an SH2 docking protein that transduces insulin's signal multidirectionally.