Ishiki M, Sasaoka T, Ishihara H, Imamura T, Usui I, Takata Y, Kobayashi M
First Department of Medicine, Toyama Medical and Pharmaceutical University, Japan.
Endocrinology. 1997 Nov;138(11):4950-8. doi: 10.1210/endo.138.11.5510.
We examined the potential role of Crk-II in insulin and epidermal growth factor (EGF) signaling in Rat-1 fibroblasts overexpressing insulin receptors. Crk is an SH2 and SH3 domain-containing adaptor protein that has been reported to associate with p130cas, paxillin, c-cbl, c-abl, Sos, and C3G in vitro. Insulin- and EGF-induced association of Crk-II with these molecules was assessed by immunoblotting of anti-Crk-II precipitates in Rat-1 fibroblasts overexpressing insulin receptors. Neither insulin nor EGF treatment induced Crk-II association with either Sos or C3G. Basal tyrosine phosphorylation of c-abl and its constitutive association with Crk-II were not further increased by insulin or EGF. p130cas and paxillin were heavily tyrosine phosphorylated in the basal state. Both insulin and EGF stimulated their dephosphorylation, followed by p130cas-Crk-II dissociation and paxillin-Crk-II association, although the magnitude of these effects was greater with insulin than with EGF. Interestingly, EGF, but not insulin, stimulated tyrosine phosphorylation of c-cbl and its association with Crk-II. To investigate the functional roles of Crk-II in mitogenesis and cytoskeletal rearrangement, we performed microinjection analysis. Cellular microinjection of anti-Crk-II antibody inhibited EGF-induced, but not insulin-induced, DNA synthesis. Insulin, but not EGF, stimulated cytoskeletal rearrangement in the cells, and microinjection of anti-Crk-II antibody effectively inhibited insulin-induced membrane ruffling, suggesting that Crk-II is involved in insulin-induced cytoskeletal rearrangement. These results indicate that Crk-II functions as a multifunctional adaptor molecule linking insulin and EGF receptors to their downstream signals. The presence of c-cbl-Crk-II association may partly determine the signal specificities initiated by insulin and EGF.
我们研究了Crk-II在过表达胰岛素受体的大鼠-1成纤维细胞中，在胰岛素和表皮生长因子（EGF）信号传导中的潜在作用。Crk是一种含有SH2和SH3结构域的衔接蛋白，据报道在体外它可与p130cas、桩蛋白、c-cbl、c-abl、Sos和C3G结合。通过对过表达胰岛素受体的大鼠-1成纤维细胞中抗Crk-II沉淀物进行免疫印迹，评估胰岛素和EGF诱导的Crk-II与这些分子的结合情况。胰岛素和EGF处理均未诱导Crk-II与Sos或C3G结合。胰岛素或EGF未进一步增加c-abl的基础酪氨酸磷酸化及其与Crk-II的组成性结合。p130cas和桩蛋白在基础状态下大量酪氨酸磷酸化。胰岛素和EGF均刺激它们的去磷酸化，随后p130cas-Crk-II解离和桩蛋白-Crk-II结合，尽管胰岛素的这些作用程度大于EGF。有趣的是，EGF而非胰岛素刺激了c-cbl的酪氨酸磷酸化及其与Crk-II的结合。为了研究Crk-II在有丝分裂和细胞骨架重排中的功能作用，我们进行了显微注射分析。细胞显微注射抗Crk-II抗体抑制了EGF诱导的而非胰岛素诱导的DNA合成。胰岛素而非EGF刺激了细胞中的细胞骨架重排，显微注射抗Crk-II抗体有效抑制了胰岛素诱导的膜皱褶，表明Crk-II参与胰岛素诱导的细胞骨架重排。这些结果表明，Crk-II作为一种多功能衔接分子，将胰岛素和EGF受体与其下游信号联系起来。c-cbl-Crk-II结合的存在可能部分决定了胰岛素和EGF引发的信号特异性。
