The activation of the protein kinase C (PKC) family of serine/ threonine kinases contributes to the modulation of insulin signaling, and the PKC-dependent phosphorylation of insulin receptor substrate (IRS)-1 has been implicated in the development of insulin resistance. Here we demonstrate Ser 357 of rat IRS-1 as a novel PKC-␦-dependent phosphorylation site in skeletal muscle cells upon stimulation with insulin and phorbol ester using Ser(P) 357 antibodies and active and kinase dead mutants of PKC-␦. Phosphorylation of this site was simulated using IRS-1 Glu 357 and shown to reduce insulin-induced tyrosine phosphorylation of IRS-1, to decrease activation of Akt, and to subsequently diminish phosphorylation of glycogen synthase kinase-3. When the phosphorylation was prevented by mutation of Ser 357 to alanine, these effects of insulin were enhanced. When the adjacent Ser To accomplish its fundamental role in maintaining in vivo metabolic homeostasis, insulin binds and activates insulin receptors, which in turn recruit insulin-receptor substrate (IRS) 2 proteins (1). By disruption of their respective genes in mice, IRS-1 and IRS-2 have been assigned a central role in mediating the normal actions of insulin. Defects at the level of the IRS proteins also comprise a major locus for the development of metabolic disorders, including insulin resistance and type 2 diabetes (2, 3). IRS proteins are regulated at several steps, including gene expression, protein degradation, and phosphorylation (4, 5). Although the phosphorylation of IRS-1 on tyrosine residues is mandatory for insulin-stimulated responses, serine/threonine phosphorylation appears to be the mechanism for the precise regulation and could either enhance or attenuate the insulin effects (6, 7). However, in most studies serine phosphorylation of IRS-1 has been implicated as a negative regulator of insulin signaling (8 -12). It can induce the dissociation of IRS-1 from its receptor and hinder the phosphorylation of tyrosine residues (8), release the IRS-1 from intracellular complexes that maintain them in close proximity to the receptor (13), or induce its protein degradation (14). These multiple effects suggest that the serine residues subjected to phosphorylation play a pivotal role in regulating IRS-1 function. Of note, the unbalanced chronic stimulation of IRS-1 serine kinases leads to hyperphosphorylation of IRS-1 and is a major pathophysiological mechanism in the development of insulin resistance. Among these IRS-1 kinases, members of the protein kinase C (PKC) family of serine/threonine kinases have received considerable attention for their regulatory role in insulin signaling. Although particularly the activation of atypical PKCs has been reported as positive modulation of insulin signaling (15, 16), the majority of studies has demonstrated that PKCs are implicated in impaired insulin signaling including classical (17-19), novel (20), and more recently also atypical PKC isoforms (21). Activation of PKC isoforms by insulin (11, 21, 22), hyperglycemia (1...