No abstract

The regulatory in¯uences of glycogen synthase kinase-3b (GSK3b) and lithium on the activity of cyclic AMP response element binding protein (CREB) were examined in human neuroblastoma SH-SY5Y cells. Activation of Akt (protein kinase B) with serum-increased phospho-serine-9-GSK3b (the inactive form of the enzyme), inhibited GSK3b activity, and increased CREB DNA binding activity. Inhibition of GSK3b by another paradigm, treatment with the selective inhibitor lithium, also increased CREB DNA binding activity. The inhibitory regulation of CREB DNA binding activity by GSK3b also was evident in differentiated SH-SY5Y cells, indicating that this regulatory interaction is maintained in nonproliferating cells. These results demonstrate that inhibition of GSK3b by serine-9 phosphorylation or directly by lithium increases CREB activation. Conversely, overexpression of active GSK3b to 3.5-fold the normal levels completely blocked increases in CREB DNA binding activity induced by epidermal growth factor, insulin-like growth factor-1, forskolin, and cyclic AMP. The inhibitory effects due to overexpressed GSK3b were reversed by treatment with lithium and with another GSK3b inhibitor, sodium valproate. Overall, these results demonstrate that GSK3b inhibits, and lithium enhances, CREB activation.

No abstract

Abbreviations used: ACPD, trans-i -aminocyclopentyl-1 ,3-dicarboxylic acid; ANOVA, analysis of variance; FC8/9 and FC1O, frontal cortex areas 8/9 and 10, respectively; GAP-43, growth-associated protein-43; GTPyS, guanosine 5 '-O-(3-thiotriphosphate); P1, phosphatidylinositol; SDS, sodium dodecyl sulfate.

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.