Chen Y, Grall D, Salcini A E, Pelicci P G, Pouysségur J, Van Obberghen-Schilling E
Centre de Biochimie, CNRS UMR134, Parc Valrose, Nice, France.
EMBO J. 1996 Mar 1;15(5):1037-44.
The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation of the thrombin receptor in growth-responsive CCL39 fibroblasts. Shc phosphorylation by thrombin or the thrombin receptor agonist peptide is maximal by 15 min and persists for > or = 2 h. Following thrombin stimulation, phosphorylated Shc is recruited to Grb2 complexes. One or more pertussis toxin-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4-beta-phorbol-12,13-dibutyrate has no effect. Rather, thrombin-induced Shc phosphorylation is enhanced in cells depleted of phorbol ester-sensitive protein kinase C isoforms. Expression of mutant Shc proteins defective in Grb2 binding displays a dominant-negative effect on thrombin-stimulated p44 MAP kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors.
丝氨酸蛋白酶凝血酶激活G蛋白信号系统，该信号系统可导致Ras激活，并且在某些细胞中还会引发增殖。虽然G蛋白偶联受体激活Ras的具体步骤尚不明确，但受体酪氨酸激酶激活Ras的机制最近已通过生化和遗传学方法得以阐明。本研究旨在确定这两类不同的受体是否使用共同的信号转导成分。在此我们报告，在生长反应性CCL39成纤维细胞中，凝血酶受体受到刺激后，衔接蛋白Shc的酪氨酸残基会发生磷酸化。凝血酶或凝血酶受体激动剂肽诱导的Shc磷酸化在15分钟时达到最大值，并持续≥2小时。凝血酶刺激后，磷酸化的Shc会被招募到Grb2复合物中。一种或多种对百日咳毒素不敏感的蛋白质似乎介导了这种效应，因为：（i）用百日咳毒素预处理细胞并不会减弱凝血酶的作用；（ii）毒蕈碱m1受体可刺激Shc酪氨酸磷酸化。Shc磷酸化似乎不涉及蛋白激酶C，因为添加4-β-佛波醇-12,13-二丁酸酯没有作用。相反，在缺乏佛波酯敏感的蛋白激酶C亚型的细胞中，凝血酶诱导的Shc磷酸化增强。在Grb2结合方面存在缺陷的突变型Shc蛋白的表达，对凝血酶刺激的p44 MAP激酶激活、基因诱导和细胞生长具有显性负效应。根据这些数据，我们得出结论，Shc代表了受体酪氨酸激酶和G蛋白偶联受体激活的信号通路之间的一个关键交汇点。
