Insulin resistance due to phosphorylation of insulin receptor substrate-1 at serine 302.
Inhibitory serine phosphorylation is a potential molecular mechanism for insulin resistance. We have developed a new variant of the yeast two-hybrid method, referred to as disruptive yeast tri-hybrid (Y3H), to identify inhibitory kinases and sites of phosphorylation in insulin receptors (IR) and IR substrates, IRS-1. Using IR and IRS-1 as bait and prey, respectively, and c-Jun NH(2)-terminal kinase (JNK1) as the disruptor, we now show that phosphorylation of IRS-1 Ser-307, a previously identified site, is necessary but not sufficient for JNK1-mediated disruption of IR/IRS-1 binding. We further identify a new phosphorylation site, Ser-302, and show that this too is necessary for JNK1-mediated disruption. Seven additional kinases potentially linked to insulin resistance similarly block IR/IRS-1 binding in the disruptive Y3H, but through distinct Ser-302- and Ser-307-independent mechanisms. Phosphospecific antibodies that recognize sequences surrounding Ser(P)-302 or Ser(P)-307 were used to determine whether the sites were phosphorylated under relevant conditions. Phosphorylation was promoted at both sites in Fao hepatoma cells by reagents known to promote Ser/Thr phosphorylation, including the phorbol ester phorbol 12-myristate 13-acetate, anisomycin, calyculin A, and insulin. The antibodies further showed that Ser(P)-302 and Ser(P)-307 are increased in animal models of obesity and insulin resistance, including genetically obese ob/ob mice, diet-induced obesity, and upon induction of hyperinsulinemia. These findings demonstrate that phosphorylation at both Ser-302 and Ser-307 is necessary for JNK1-mediated inhibition of the IR/IRS-1 interaction and that Ser-302 and Ser-307 are phosphorylated in parallel in cultured cells and in vivo under conditions that lead to insulin resistance.
Werner
Eric D
ED
Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.
Lee
Jongsoon
J
Hansen
Lone
L
Yuan
Minsheng
M
Shoelson
Steven E
SE
D000818:Animals; D006946:Hyperinsulinism / Q000209:etiology / Q000378:metabolism; D055504:Insulin Receptor Substrate Proteins; D007333:Insulin Resistance; D051379:Mice; D008820:Mice, Obese; D009765:Obesity / Q000209:etiology / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism; D011972:Receptor, Insulin / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D012694:Serine; D020798:Two-Hybrid System Techniques
D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; D010750:Phosphoproteins; D016755:Proto-Oncogene Proteins c-jun; D012694:Serine; D011972:Receptor, Insulin