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

Liberman Z, Plotkin B, Tennenbaum T, Eldar-Finkelman H. Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase C II in the diabetic fat tissue. Am J Physiol Endocrinol Metab 294: E1169–E1177, 2008. First published April 22, 2008; doi:10.1152/ajpendo.00050.2008.— 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. However, the fact that GSK-3 requires prephosphorylation of its substrates suggested that Ser 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 Ser and Ser in physiologically relevant animal models. Of several stimulators, only the PKC activator phorbol ester PMA enhanced IRS-1 phosphorylation at Ser. Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKC or PKC II isoforms in cells enhanced IRS-1 phosphorylation at Ser and Ser, and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at Ser. The expression level and activation state of PKC II, but not PKC , 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 PKC II were also associated with enhanced phosphorylation of IRS-1 at Ser and elevated activity of GSK-3 . Finally, adenoviral mediated expression of PKC II in adipocytes enhancedphosphorylation of IRS-1 at Ser. Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKC II and GSK-3 at Ser and Ser. Furthermore, these data provide evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in fat tissue.