Oxidative stress oppositely modulates protein tyrosine phosphorylation stimulated by muscarinic G protein-coupled and epidermal growth factor receptors

This study's goals were to more fully define the activation of protein tyro sine phosphorylation stimulated by muscarinic receptors, to test if this si gnaling process is affected by oxidative stress induced by H2O2, and to com pare the effects of H2O2 on protein tyrosine phosphorylation activated by e pidermal growth factor (EGF) receptors, Experiments used human neuroblastom a SH-SY5Y cells which express endogenous M3 muscarinic and EGF receptors, C arbachol induced time-dependent increases in phosphotyrosine immunoreactivi ty of several protein bands, which were quantitated, and immunoprecipitatio n was used to identify the adhesion-related proteins focal adhesion kinase, p130Cas/HEF1, and paxillin, and three shc adapter proteins. Carbachol-indu ced tyrosine phosphorylation of the adhesion-related proteins was mediated by muscarinic receptors, and was inhibited by a src family kinase inhibitor , PP1, That carbachol can activate src family kinases was indicated further by the finding that carbachol induced an increase in tyrosine phosphorylat ion of p120-src substrate, which was inhibited by PP1, Oxidative stress ind uced by H2O2 concentration dependently inhibited carbachol-induced tyrosine phosphorylation of each of the adhesion-related proteins. EGF increased th e phosphotyrosine immunoreactivity of 180- and 116-kDa proteins, identified as the EGF receptor and Cbl, respectively. In contrast to the results with carbachol, H2O2 potentiated EGF-induced tyrosine phosphorylation, These re sults demonstrate that muscarinic receptor activation induces previously un recognized increases in tyrosine phosphorylation, and that this signaling p rocess is impaired by H2O2, whereas protein tyrosine phosphorylation stimul ated by EGF is increased by H2O2. Thus, oxidative stress can oppositely mod ulate protein tyrosine phosphorylation induced by activation of G protein-c oupled and growth factor receptors in the same cells. J, Neurosci. Res. 55: 329-340, 1999. (C) 1999 Wiley-Liss, Inc.