Ichiba T, Kuraishi Y, Sakai O, Nagata S, Groffen J, Kurata T, Hattori S, Matsuda M
Department of Pathology, National Institute of Health, Shinjuku-ku, Tokyo 162, Japan.
J Biol Chem. 1997 Aug 29;272(35):22215-20. doi: 10.1074/jbc.272.35.22215.
Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1 in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.
Crk是一种几乎完全由SH2和SH3结构域组成的衔接蛋白。我们之前通过体内和体外系统证明，被鉴定为Crk SH3结构域结合鸟嘌呤核苷酸交换因子的C3G可特异性激活Rap1。C3G还与其他衔接蛋白结合，包括CrkL和Grb2。在本研究中，我们分析了Crk、CrkL和Grb2对C3G-Rap1信号通路的影响。在Cos1细胞中，Crk、CrkL和Grb2与C3G共表达显著增加了与Rap1结合的GTP/GDP比例。Crk的SH2和SH3结构域对于此活性均是必需的。然而，Crk在体外并未刺激C3G对Rap1的鸟嘌呤核苷酸交换活性，这表明Crk并非通过变构机制激活C3G。向Crk添加法尼基化信号可补偿Crk的SH2结构域对Rap1鸟嘌呤核苷酸交换活性增强的需求，这表明Crk通过将C3G募集到细胞膜来增强C3G的鸟嘌呤核苷酸交换活性。这些结果表明，Crk、CrkL和Grb2主要通过将C3G募集到细胞膜来正向调节C3G-Rap1信号通路。
