Tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) by oxidant stress in cerebellar granule neurons: modulation by N-methyl-D-aspartate through calcineurin activity.

Insulin receptor-substrate-1 (IRS-1) is a docking protein for several tyrosine kinase receptors. Upon tyrosine phosphorylation, IRS-1 binds to signaling molecules that express Src homology 2 (SH-2) binding domains, including phosphatidylinositol 3-kinase (PI 3-kinase), phosphotyrosine phosphatase SHP-2 (Syp), Nck, Crk and Grb-2. Hydrogen peroxide (H(2)O(2)) induces tyrosine phosphorylation of key signaling mediators presumably by inhibition of tyrosine phosphatases. In many cell types, the activation of extracellular signal-related kinases (e.g. MAPK) and other protein kinases by H(2)O(2) leads to transcriptional activation. In the current study, we examined the effect of H(2)O(2) on IRS-1 tyrosine phosphorylation in primary cultured rat cerebellar granule neurons. H(2)O(2) stimulated the rapid tyrosine phosphorylation of IRS-1 and p42/p44 MAP kinase, and induced its association with PI 3-kinase. H(2)O(2)-induced IRS-1 phosphorylation was rapidly reversible (5 min) whereas MAPK phosphorylation persisted for up to 1 h. NMDA reversed H(2)O(2)-mediated tyrosine phosphorylation of IRS-1 and its association with PI 3-kinase. The dephosphorylation of IRS-1 by NMDA was calcium-dependent and was inhibited by the calcineurin inhibitor cyclosporine. Calmodulin-dependent tyrosine phosphatase activity of calcineurin was observed in vitro using both immunoprecipitated and recombinant tyrosine-phosphorylated IRS-1 as substrates. These data highlight the role of multiple phosphatases in the regulation of IRS-1 tyrosine phosphorylation and identify a novel functional property of calcineurin.