MUTANT OF INSULIN-RECEPTOR SUBSTRATE-1 INCAPABLE OF ACTIVATING PHOSPHATIDYLINOSITOL 3-KINASE DID NOT MEDIATE INSULIN-STIMULATED MATURATION OF XENOPUS-LAEVIS OOCYTES

Insulin receptor substrate-1 (IRS-1) is rapidly phosphorylated on mult iple tyrosine residues in response to insulin and binds several Src ho mology 2 domain-containing proteins, thereby initiating downstream sig naling. To assess the tyrosine phosphorylation sites that mediate rele vant downstream signaling and biological effects, we created site-dire cted mutants of IRS-1 and overexpressed them in the Xenopus laevis ooc yte. In oocytes overexpressing IRS-1 or IRS-1-895F (Tyr-895 replaced w ith phenylalanine), insulin activated phosphatidylinositol (PI) 3-kina se, p70 S6 kinase, and mitogen-activated protein kinase and induced oo cyte maturation. In contrast, in oocytes overexpressing IRS-1-4F (Tyr- 460, Tyr-608, Tyr-939, and Tyr-987 of IRS-1 replaced with phenylalanin e), insulin did not activate PI 3-kinase, p70 S6 kinase, and mitogen-a ctivated protein kinase and failed to induce oocyte maturation. These observations indicate that in X. laevis oocytes overexpressing IRS-1, the association of PI 3-kinase rather than Grb2 (growth factor-bound p rotein 2) with IRS-1 plays a major role in insulin induced oocyte matu ration. Activation of PI 3-kinase may lie upstream of mitogen-activate d protein kinase activation and p70 S6 kinase activation in response t o insulin.