A novel 68-kDa adipocyte protein phosphorylated on tyrosine in response toinsulin and osmotic shock

Osmotic shock can cause insulin resistance in 3T3-L1 adipocytes by inhibiti ng insulin activation of glucose transport, p70S6 kinase, glycogen synthesi s, and lipogenesis. By further investigating the relationship between insul in and hypertonic stress, we have discovered that osmotic shock enhanced by 10-fold the insulin-stimulated tyrosine phosphorylation of a 68-hDa protei n. Phosphorylation by insulin was maximal after 1 min and was saturated wit h 50-100 nM insulin. The effect of sorbitol was completely reversible by 2. 5 min. pp68 was a peripheral protein that was localized to the detergent in soluble fraction of the low density microsomes but was not associated with the cytoskeleton. Stimulation of the p42/44 and the p38 MAP kinase pathways by osmotic shock had no effect on pp68 phosphorylation. Treatment of adipo cytes with the phosphotyrosine phosphatase inhibitor phenylarsine oxide als o enhanced insulin-activated tyrosine phosphorylation of pp68 suggesting th at osmotic shock may increase pp68 phosphorylation by inhibiting a phosphot yrosine phosphatase. Dissociation of pp68 from the low density microsomes w ith RNase A indicated that pp68 binds to RNA. Failure to immunoprecipitate pp68 using antibodies directed against known 60-70-kDa tyrosine-phosphoryla ted proteins suggest that pp68 may be a novel cellular target that lies dow nstream of the insulin receptor.