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 tyros ine kinase receptors. Upon tyrosine phosphorylation, IRS-1 binds to Signali ng molecules that express Src homology 2 (SH-2) binding domains, including phosphatidylinositol 3-kinase (Pl 3-kinase), phosphotyrosine phosphatase SH P-2 (Syp), Nck, Crk and Grb-2. Hydrogen peroxide (H2O2) induces tyrosine ph osphorylation of key signaling mediators presumably by inhibition of tyrosi ne phosphatases. In many cell types, the activation of extracellular signal -related kinases e.g. MAPK) and other protein kinases by H2O2 leads to tran scriptional activation. In the current study, we examined the effect of H2O 2 on IRS-1 tyrosine phosphorylation in primary cultured rat cerebellar gran ule neurons. H2O2 stimulated the rapid tyrosine phosphorylation of IRS-1 an d p42/p44 MAP kinase, and induced its association with PI 3-kinase. H2O2-in duced IRS-1 phosphorylation was rapidly reversible (5 min) whereas MAPK pho sphorylation persisted for up to 1 h. NMDA reversed H2O2-mediated tyrosine phosphorylation of IRS-1 and its association with Pl 3-kinase. The dephosph orylation of IRS-1 by NMDA was calcium-dependent and was inhibited by the c alcineurin inhibitor cyclosporine. Calmodulin-dependent tyrosine phosphatas e activity of calcineurin was observed in vitro using both immunoprecipitat ed and recombinant tyrosine-phosphorylated IRS-1 as substrates. These data highlight the role of multiple phosphatases in the regulation of IRS-1 tyro sine phosphorylation and identify a novel functional property of calcineuri n.