Serine Phosphorylation of Insulin Receptor Substrate 1 by Inhibitor κB Kinase Complex* 210

Insulin resistance contributes importantly to the pathophysiology of type 2 diabetes mellitus. One mechanism mediating insulin resistance may involve the phosphorylation of serine residues in insulin receptor substrate-1 (IRS-1), leading to impairment in the ability of IRS-1 to activate downstream phosphatidylinositol 3-kinase-dependent pathways. Insulin-resistant states and serine phosphorylation of IRS-1 are associated with the activation of the inhibitor κB kinase (IKK) complex. However, the precise molecular mechanisms by which IKK may contribute to the development of insulin resistance are not well understood. In this study, using phosphospecific antibodies against rat IRS-1 phosphorylated at Ser307 (equivalent to Ser312 in human IRS-1), we observed serine phosphorylation of IRS-1 in response to TNF-α or calyculin A treatment that paralleled surrogate markers for IKK activation. The phosphorylation of human IRS-1 at Ser312 in response to tumor necrosis factor-α was significantly reduced in cells pretreated with the IKK inhibitor 15 deoxy-prostaglandin J2 as well as in cells derived from IKK knock-out mice. We observed interactions between endogenous IRS-1 and IKK in intact cells using a co-immunoprecipitation approach. Moreover, this interaction between IRS-1 and IKK in the basal state was reduced upon IKK activation and increased serine phosphorylation of IRS-1. Data from in vitro kinase assays using recombinant IRS-1 as a substrate were consistent with the ability of IRS-1 to function as a direct substrate for IKK with multiple serine phosphorylation sites in addition to Ser312. Taken together, our data suggest that IRS-1 is a novel direct substrate for IKK and that phosphorylation of IRS-1 at Ser312 (and other sites) by IKK may contribute to the insulin resistance mediated by activation of inflammatory pathways.

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