Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase C betaII 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 Ser(336) 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(336) and Ser(332) in physiologically relevant animal models. Of several stimulators, only the PKC activator phorbol ester PMA enhanced IRS-1 phosphorylation at Ser(336). Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKCalpha or PKCbetaII isoforms in cells enhanced IRS-1 phosphorylation at Ser(336) and Ser(332), and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at Ser(336). The expression level and activation state of PKCbetaII, but not PKCalpha, 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 PKCbetaII were also associated with enhanced phosphorylation of IRS-1 at Ser(336/332) and elevated activity of GSK-3beta. Finally, adenoviral mediated expression of PKCbetaII in adipocytes enhancedphosphorylation of IRS-1 at Ser(336). Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKCbetaII and GSK-3 at Ser(336) and Ser(332). Furthermore, these data provide evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in fat tissue.
Liberman
Ziva
Z
Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel.
Plotkin
Batya
B
Tennenbaum
Tamar
T
Eldar-Finkelman
Hagit
H
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000273:Adipose Tissue / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002070:Butadienes / Q000494:pharmacology; D016466:CHO Cells; D002227:Carbazoles / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology / Q000378:metabolism*; D003924:Diabetes Mellitus, Type 2 / Q000201:enzymology / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008820:Mice, Obese; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D064546:Protein Kinase C beta; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; D002070:Butadienes; D002227:Carbazoles; D007211:Indoles; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D008301:Maleimides; D009570:Nitriles; D047428:Protein Kinase Inhibitors; C113580:U 0126; C081021:Go 6976; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D038362:Glycogen Synthase Kinase 3; C070515:bisindolylmaleimide I