Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase CβII in the diabetic fat tissue

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is an important negative modulator of insulin signaling. Previously, we showed that glycogen synthase kinase-3 (GSK-3) phosphorylates IRS-1 at Ser ³³². However, the fact that GSK-3 requires prephosphorylation of its substrates suggested that Ser³³⁶ on IRS-1 was the "priming" site phosphorylated by an as yet unknown protein kinase. Here, we sought to identify this "priming kinase" and to examine the phosphorylation of IRS-1 at Ser³³⁶ and Ser³³² in physiologically relevant animal models. Of several stimulators, only the PKC activator phorbol ester PMA enhanced IRS-1 phosphorylation at Ser³³⁶. Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKCα or PKCβII isoforms in cells enhanced IRS-1 phosphorylation at Ser³³⁶ and Ser³³², and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at Ser³³⁶. The expression level and activation state of PKCβII, but not PKCα, were remarkably elevated in the fat tissues of diabetic ob/ob mice and in high-fat diet-fed mice compared with that from lean animals. Elevated levels of PKCβII were also associated with enhanced phosphorylation of IRS-1 at Ser^336/332 and elevated activity of GSK-3β. Finally, adenoviral mediated expression of PKCβII in adipocytes enhancedphosphorylation of IRS-1 at Ser³³⁶. Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKCβII and GSK-3 at Ser³³⁶ and Ser³³². Furthermore, these data provide evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in fat tissue.