Ser/Thr phosphorylation of insulin receptor substrate IRS-1 regulates insulin signaling, but the relevant phosphorylated residues and their potential functions during insulin-stimulated signal transduction are difficult to resolve. We used a sequence-specific polyclonal antibody directed against phosphorylated Ser 302 to study IRS-1-mediated signaling during insulin and insulinlike growth factor IGF-I stimulation. Many insulin signals are produced through tyrosine phosphorylation of the insulin receptor substrate (IRS) 1 -proteins or other scaffold proteins, including Shc, Cbl, APS, and SH2B, Gab1/2, and Dock1/2 (1). Although the role of each of these substrates merits attention, work with transgenic mice suggests that many insulin responses, especially those that are associated with somatic growth and carbohydrate metabolism, are mediated through IRS-1 or IRS-2 (2-4). IRS-proteins are composed of multiple interaction domains and phosphorylation motifs, but they appear to lack intrinsic catalytic activities (5). All IRS-proteins contain an NH 2 -terminal pleckstrin homology domain followed by a phosphotyrosine binding domain. These domains couple the IRS-protein to the activated receptors for insulin, IGF-I, IL-4 and others (6). Multiple tyrosine phosphorylation sites in the COOH terminus recruit various enzymes (PI3K, SHP2, or fyn) or adapter proteins (Grb-2, nck, crk, shb, and others) that promote carbohydrate and lipid metabolism, cell growth and survival, and the function of specialized neuroendocrine tissues that promote nutrient homeostasis and female fertility (5, 7).Insulin plays a pivotal role in metabolic regulation because it promotes nutrient influx and storage in muscle and adipocytes and inhibits hepatic gluconeogenesis. Although insulin is the principal activator, heterologous signals reflecting nutrient availability or physiological stress modulate insulin signaling. Dysregulation of these heterologous signals contributes to peripheral insulin resistance, metabolic dysregulation, and diabetes (2). Several mechanisms have been proposed to explain heterologous regulation of the insulin signaling cascade, including cytokine-induced serine phosphorylation or degradation of the IRS-proteins, or direct inhibition of the insulin receptor. IRS-1 contains more than 20 potential serine phosphorylation sites in amino acid sequence motifs that are recognized by various kinases, including protein kinase A, protein kinase B/Akt, protein kinase C, ERK1/2, Rho kinase and mTOR (8 -10). Many studies suggest that serine phosphorylation causes insulin resistance by blocking the interaction between IRS-1 and the activated insulin receptor, inhibiting the phosphorylation of specific tyrosine residues, or promoting IRS-1 degradation (11-13). Previous work from our laboratory shows that JNK-mediated phosphorylation of Ser 307 inhibits coupling of IRS-1 to the activated insulin receptor, which reduces insulin-stimulated tyrosine phosphorylation and insulin signaling (14,15). By contrast, here we show that an adjacent r...
