Jope R S, Song L, Grimes C A, Zhang L
Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, 35294-0017, USA.
J Neurosci Res. 1999 Feb 1;55(3):329-40. doi: 10.1002/(SICI)1097-4547(19990201)55:3<329::AID-JNR8>3.0.CO;2-K.
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.
本研究的目的是更全面地定义毒蕈碱受体刺激引起的蛋白酪氨酸磷酸化激活，测试该信号传导过程是否受H2O2诱导的氧化应激影响，并比较H2O2对表皮生长因子（EGF）受体激活的蛋白酪氨酸磷酸化的影响。实验使用表达内源性M3毒蕈碱受体和EGF受体的人神经母细胞瘤SH-SY5Y细胞。卡巴胆碱诱导几条蛋白条带的磷酸酪氨酸免疫反应性呈时间依赖性增加，对其进行了定量分析，并采用免疫沉淀法鉴定了与黏附相关的蛋白——粘着斑激酶、p130Cas/HEF1和桩蛋白，以及三种shc衔接蛋白。卡巴胆碱诱导的与黏附相关蛋白的酪氨酸磷酸化由毒蕈碱受体介导，并被src家族激酶抑制剂PP1抑制。卡巴胆碱可激活src家族激酶这一点进一步通过以下发现得到证实：卡巴胆碱诱导p120-src底物的酪氨酸磷酸化增加，而这被PP1抑制。H2O2诱导的氧化应激浓度依赖性地抑制了卡巴胆碱诱导的每种与黏附相关蛋白的酪氨酸磷酸化。EGF增加了180 kDa和116 kDa蛋白的磷酸酪氨酸免疫反应性，分别鉴定为EGF受体和Cbl。与卡巴胆碱的结果相反，H2O2增强了EGF诱导的酪氨酸磷酸化。这些结果表明，毒蕈碱受体激活诱导了先前未被认识到的酪氨酸磷酸化增加，并且该信号传导过程被H2O2损害，而EGF刺激的蛋白酪氨酸磷酸化则被H2O2增强。因此，氧化应激可对同一细胞中G蛋白偶联受体和生长因子受体激活诱导的蛋白酪氨酸磷酸化产生相反的调节作用。
