Werner, Eric D.Lee, JongsoonHansen, LoneYuan, MinshengShoelson, Steven E.2019-10-172004Werner, Eric D., Jongsoon Lee, Lone Hansen, Minsheng Yuan, and Steven E. Shoelson. 2004. “Insulin Resistance Due to Phosphorylation of Insulin Receptor Substrate-1 at Serine 302.” Journal of Biological Chemistry 279 (34). American Society for Biochemistry & Molecular Biology (ASBMB): 35298–305. doi:10.1074/jbc.m405203200.0021-92581083-351Xhttp://nrs.harvard.edu/urn-3:HUL.InstRepos:41555765Inhibitory 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 NH2-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.en-USJournal Article2019-10-1710.1074/jbc.M405203200