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 tyrosine phosphorylation stimulated by muscarinic receptors, to test if this signaling process is affected by oxidative stress induced by H₂O₂, and to compare the effects of H₂O₂ on protein tyrosine phosphorylation activated by epidermal growth factor (EGF) receptors. Experiments used human neuroblastoma SH-SY5Y cells which express endogenous M3 muscarinic and EGF receptors. Carbachol induced time-dependent increases in phosphotyrosine immunoreactivity of several protein bands, which were quantitated, and immunoprecipitation was used to identify the adhesion-related proteins focal adhesion kinase, p130Cas/HEF1, and paxillin, and three shc adapter proteins. Carbachol-induced 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 phosphorylation of p120-src substrate, which was inhibited by PP1. Oxidative stress induced by H₂O₂ concentration dependently inhibited carbachol-induced tyrosine phosphorylation of each of the adhesion-related proteins. EGF increased the 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 results demonstrate that muscarinic receptor activation induces previously unrecognized increases in tyrosine phosphorylation, and that this signaling process is impaired by H₂O₂, whereas protein tyrosine phosphorylation stimulated by EGF is increased by H₂O₂. Thus, oxidative stress can oppositely modulate protein tyrosine phosphorylation induced by activation of G protein-coupled and growth factor receptors in the same cells.