Kobayashi S, Okumura N, Okada M, Nagai K
Division of Protein Metabolism, Institute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita, Osaka 565.
J Biochem. 1998 Apr;123(4):624-9. doi: 10.1093/oxfordjournals.jbchem.a021983.
KCl-treatment of PC12 cells induces depolarization of the plasma membrane and Ca2+ influx into the cells. We have previously shown that KCl induced tyrosine phosphorylation of cellular proteins of 120, 110, 68, 44, and 42 k, and that the 68 k protein was paxillin. In the present study, we found that the 120 k protein was a Crk-associated Src substrate, p130(cas). KCl-induced tyrosine phosphorylation of p130(cas) was not observed in EGTA-containing medium, suggesting that it was due to Ca2+ influx into the cells. Time course experiments showed that tyrosine phosphorylation of p130(cas) peaked at 5 min after stimulation and returned to the basal level at 60 min, while mobility shift of p130(cas) was observed within 2 min and lasted over 60 min, indicating that serine or threonine residues, in addition to tyrosine, were phosphorylated on KCl stimulation. In vitro kinase assay of immunoprecipitates with anti-p130(cas) antibody suggested that some protein-tyrosine kinases were associated with p130(cas). Using the substrate region of p130(cas) as the substrate, we found that Fyn and Src were activated on stimulation with KCl. These results indicate that tyrosine phosphorylation of p130(cas) may be involved in Ca2+-dependent events in neuronal and neuroendocrine cells.
用氯化钾处理PC12细胞可诱导质膜去极化以及钙离子流入细胞。我们之前已经表明，氯化钾可诱导120k、110k、68k、44k和42k的细胞蛋白发生酪氨酸磷酸化，并且68k的蛋白是桩蛋白。在本研究中，我们发现120k的蛋白是一种与Crk相关的Src底物，即p130(cas)。在含有乙二醇双乙醚二胺四乙酸（EGTA）的培养基中未观察到氯化钾诱导的p130(cas)酪氨酸磷酸化，这表明其是由于钙离子流入细胞所致。时间进程实验表明，p130(cas)的酪氨酸磷酸化在刺激后5分钟达到峰值，并在60分钟时恢复到基础水平，而p130(cas)的迁移率变化在2分钟内即可观察到，并持续超过60分钟，这表明除酪氨酸外，丝氨酸或苏氨酸残基在氯化钾刺激下也会发生磷酸化。用抗p130(cas)抗体对免疫沉淀物进行体外激酶分析表明，一些蛋白酪氨酸激酶与p130(cas)相关。以p130(cas)的底物区域为底物，我们发现用氯化钾刺激时Fyn和Src被激活。这些结果表明，p130(cas)的酪氨酸磷酸化可能参与神经元和神经内分泌细胞中依赖钙离子的事件。
