Insulin-induced activation of phosphatidylinositol (PI) 3-kinase. Insulin-induced phosphorylation of insulin receptors and insulin receptor substrate-1 displaces phosphorylated platelet-derived growth factor receptors from binding sites on PI 3-kinase.

Phosphatidylinositol (PI) 3-kinase is an enzyme that functions in the signaling pathways downstream from multiple cell surface receptors. The p85 regulatory subunit of PI 3-kinase binds to phosphotyrosine residues of various phosphoproteins including the platelet-derived growth factor (PDGF) receptor, the insulin receptor, and insulin receptor substrate-1 (IRS-1). Using NIH-3T3 cells overexpressing the human insulin receptor, we demonstrate that the p85 regulatory subunit of PI 3-kinase binds to phosphorylated PDGF receptor in cells incubated in the absence of insulin. When insulin is added, p85 is released from phosphorylated PDGF receptors and binds to phosphorylated insulin receptors and insulin receptor substrate-1. Moreover, insulin-induced dissociation of PDGF receptors from binding sites on PI 3-kinase requires a functional insulin receptor and is not prevented by vanadate treatment. In contrast, insulin activation does not displace PDGF receptors from binding sites on Ras GTPase-activating protein. This competition for binding to PI 3-kinase provides a mechanism for cross-talk among signaling pathways initiated by distinct peptide hormones and growth factors such as insulin and PDGF.

Insulin-induced activation of phosphatidyl inositol 3-kinase. Demonstration that the p85 subunit binds directly to the COOH terminus of the insulin receptor in intact cells.

Insulin activates the insulin receptor tyrosine kinase to phosphorylate signaling molecules such as insulin receptor substrate-1 (IRS-1). Phosphorylated IRS-1 binds to SH2 domains in the p85 regulatory subunit of phosphatidyl inositol (PI) 3-kinase, thereby stimulating the catalytic activity of PI 3-kinase. For most growth factor receptor tyrosine kinases (including receptors for epidermal growth factor and platelet-derived growth factor), the p85 regulatory subunit of PI 3-kinase binds directly to phosphorylated YXXM motifs contained in the cytoplasmic domain of the receptor itself. Previous studies in cell-free systems have shown that the phosphorylated YHTM sequence (amino acid residues 1322-1325) in the COOH terminus of the insulin receptor has the ability to bind to the p85 subunit of PI 3-kinase, thereby activating the enzyme. In this investigation, we demonstrate the occurrence of the same direct binding interaction in intact cells. Subsequent to insulin-stimulated phosphorylation of the insulin receptor, a complex is formed that contains the insulin receptor and PI 3-kinase. This complex can be immunoprecipitated by antibodies directed against either the insulin receptor or the p85 subunit of PI 3-kinase. The delta 43 mutant insulin receptor that lacks 43 amino acids at the COOH terminus does not bind p85. In addition, the delta 43 truncation impairs the ability of the receptor to mediate the activation of PI 3-kinase. Thus, by binding directly to p85, the phosphorylated YHTM motif in the COOH terminus of the insulin receptor contributes partially to mediating the effect of insulin to activate PI 3-kinase.