Phosphatidylinositol 3-kinase (PI 3-kinase) is stimulated by insulin and a variety of growth factors, but its exact role in signal transduction remains unclear. We have used a novel, highly specific inhibitor of PI 3-kinase to dissect the role of this enzyme in insulin action. Treatment of intact 3T3-L1 adipocytes with LY294002 produced a dose-dependent inhibition of insulin-stimulated PI 3-kinase (50% inhibitory concentration, 6 t.M) with >95% reduction in the levels of phosphatidylinositol-3,4,5-trisphosphate without changes in the levels of phosphatidylinositol-4-monophosphate or its derivatives. In parallel, there was a complete inhibition of insulin-stimulated phosphorylation and activation of pp7O S6 kinase. Inhibition of PI 3-kinase also effectively blocked insulin-and serum-stimulated DNA synthesis and insulin-stimulated glucose uptake by inhibiting translocation of GLUT 4 glucose transporters to the plasma membrane. By contrast, LY294002 had no effect on insulin stimulation of mitogen-activated protein kinase or pp9O S6 kinase. Thus, activation of PI 3-kinase plays a critical role in mammalian cells and is required for activation of pp7O S6 kinase and DNA synthesis and certain forms of intracellular vesicular trafficking but not mitogen-activated protein kinase or pp9O S6 kinase activation. These data suggest that PI 3-kinase is not only an important component but also a point of divergence in the insulin signaling network.Insulin-mediated signals regulate a variety of intracellular growth and metabolic events including the uptake and disposal of glucose, transport of amino acids, transcription of specific genes, and synthesis of DNA (22,35). These signals are initiated by insulin binding to and activating its cell surface receptor tyrosine kinase, resulting in autophosphorylation of the receptor on tyrosine residues and rapid phosphorylation of an immediate downstream substrate molecule, insulin receptor substrate 1 (IRS-1) (25, 55). IRS-1 is a high-molecular-weight cytosolic protein which contains 20 potential tyrosine phosphorylation sites and over 40 potential serine/threonine phosphorylation sites and has been shown to be a major substrate for both the insulin and IGF-1 receptors (32,48). Several of the potential tyrosine phosphorylation sites reside in peptide sequences that are known to associate with proteins containing SH2 domains (48). In its phosphorylated form, IRS-1 has been shown to act as a docking protein that forms a signaling complex with phosphatidylinositol 3-kinase (PI 3-kinase), the phosphotyrosine phosphatase SHPTP2 (Syp), and GRB-2, a molecule which links IRS-1 to the p2lras signaling system (31,44,48). Association of IRS-1 with PI 3-kinase and other proteins containing SH2 domains results in their activation, and IRS-1 thus plays a pivotal role in the complex divergent network encompassing insulin's pleiotropic effects (2,33,41).In addition to its interaction with IRS-1, PI 3-kinase directly associates with and is stimulated by a number of activated growth factor receptors and n...