Okada S, Pessin J E
Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242, USA.
J Biol Chem. 1997 Nov 7;272(45):28179-82. doi: 10.1074/jbc.272.45.28179.
Insulin and epidermal growth factor (EGF) stimulation of Chinese hamster ovary cells expressing the human insulin and EGF receptors resulted in a time-dependent decrease in the ability of a Rap1 antibody (amino acid epitope 121-136) to immunoprecipitate Rap1 from whole cell detergent extracts. This was due to an apparent masking of Rap1 as heat denaturation of the whole cell detergent extracts (5 min at 100 degrees C) resulted in equal immunoprecipitation of Rap1 with this epitope-specific antibody. The time-dependent change in Rap1 immunoreactivity was paralleled with an insulin-stimulated dissociation of the CrkII-C3G complex. Similarly, EGF treatment also resulted in a time-dependent dissociation of the CrkII-C3G complex that occurred concomitant with the masking of the 121-136 Rap1 epitope. Furthermore, pretreatment of the cells with the tyrosine kinase inhibitor, genistein, decreased both the basal and insulin-stimulated tyrosine phosphorylation of CrkII that directly correlated with the amount of CrkII that was immunoprecipitated with C3G. Together, these data suggest that insulin and EGF stimulation result in the dissociation of the CrkII-C3G complex, thereby inducing an apparent conformation change in Rap1.
用胰岛素和表皮生长因子（EGF）刺激表达人胰岛素受体和EGF受体的中国仓鼠卵巢细胞，结果显示，Rap1抗体（氨基酸表位121 - 136）从全细胞去污剂提取物中免疫沉淀Rap1的能力随时间呈下降趋势。这是由于Rap1明显被掩盖，因为全细胞去污剂提取物经热变性（100℃ 5分钟）后，该表位特异性抗体对Rap1的免疫沉淀量相等。Rap1免疫反应性随时间的变化与胰岛素刺激的CrkII - C3G复合物解离平行。同样，EGF处理也导致CrkII - C3G复合物随时间解离，同时121 - 136 Rap1表位被掩盖。此外，用酪氨酸激酶抑制剂染料木黄酮预处理细胞，可降低CrkII的基础酪氨酸磷酸化水平以及胰岛素刺激的酪氨酸磷酸化水平，这与用C3G免疫沉淀的CrkII量直接相关。这些数据共同表明，胰岛素和EGF刺激导致CrkII - C3G复合物解离，从而诱导Rap1出现明显的构象变化。
