Common Inhibitory Serine Sites Phosphorylated by IRS-1 Kinases, Triggered by Insulin and Inducers of Insulin Resistance

The Insulin Receptor Substrate (IRS) proteins are key players in insulin signal transduction and are the best studied targets of the insulin receptor. Ser/Thr phosphorylation of IRS proteins negatively modulates insulin signaling; therefore, the identification of IRS kinases and their target Ser phosphorylation sites is of physiological importance. Here we show that in Fao rat hepatoma cells, the IκB kinase β (IKKβ) is an IRS-1 kinase activated by selected inducers of insulin resistance, including sphingomyelinase, ceramide, and free fatty acids. Moreover, IKKβ shares a repertoire of seven potential target sites on IRS-1 with protein kinase C ζ (PKCζ), an IRS-1 kinase activated both by insulin and by inducers of insulin resistance. We further show that mutation of these seven sites (Ser-265, Ser-302, Ser-325, Ser-336, Ser-358, Ser-407, and Ser-408) confers protection from the action of IKKβ and PKCζ when they are overexpressed in Fao cells or primary hepatocytes. This enables the mutated IRS proteins to better propagate insulin signaling. These findings suggest that insulin-stimulated IRS kinases such as PKCζ overlap with IRS kinases triggered by inducers of insulin resistance, such as IKKβ, to phosphorylate IRS-1 on common Ser sites.