Insulin Resistance Due to Phosphorylation of IRS-1 at Serine 302

Insulin Resistance Due to Phosphorylation of IRS-1 at Serine 302 Inhibitory serine phosphorylat Inhibitory serine phosphorylation is a potential molecular mechanism for insulin resistance. We have developed a new variant of the yeast 2-hybrid method, referred to as disruptive yeast tri-hybrid (Y3H), to identify inhibitory kinases and sites of phosphorylation in insulin receptors (IR) and IRS-1. Using IR and IRS-1 as bait and prey, respectively, and JNK1 as the disruptor, we now show that phosphorylation of IRS-1 S307, 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, S302, 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, S302- and S307-independent mechanisms. Phosphospecific antibodies that recognize sequences surrounding pS302 or pS307 were used to determine whether the sites were phosphorylated under relevant conditions. Interestingly, efficient S307 phosphorylation appears to require prior phosphorylation at S302. Phosphorylation was promoted at both sites in Fao hepatoma cells by reagents known to promote S/T phosphorylation, including the phorbol ester PMA, anisomycin, calyculin A, and insulin. The antibodies further showed that pS302 and pS307 are increased in animal models of obesity and insulin resistance, including genetically obese [italic]ob/ob[/italic] mice, diet-induced obesity, and upon induction of hyperinsulinemia. These findings suggest that S302 and S307 phosphorylation are both required for JNK1-mediated inhibition of IR/IRS-1 interactions, that S302 is phosphorylated in cultured cells and [italic]in vivo[/italic] under conditions of insulin resistance, and that phosphorylations at S302 and S307 occur sequentially and under parallel conditions. JONGSOON LEE, ERIC D. WERNER, LONE HANSEN, MINSHENG YUAN, STEVEN E. SHOELSON 1289-P Boston, MA Insulin Action - Insulin Resistance