Structural study on the mechanism of the signal transduction mediated by the Ras protein

到目前为止，GAP/NF1家族、Raf家族、PI3K、RalGDS等已被鉴定出几种不同类型的依赖GTP的RAS结合域，但它们的序列同源性尚未阐明。突变的RAS蛋白具有与这些靶蛋白的子集的结合特异性，有望成为分析复杂的RAS信号通路的有用工具。在本研究中，我们使Ha-RAS突变体(S)在残基21-71区域携带取代，并测试了它们与不同RAS结合域的结合能力。实际上，我们发现了几个能与有限范围的靶标结合的突变体。核磁共振分析表明，这些突变体中的一些没有表现出野生型RAS蛋白的GTP结合形式所表现出的“区域多维现象”。因此，我们认为RAS的“区域多面性”允许与不同的靶分子结合。事实上，我们发现RAS蛋白在与Raf-1或RGL的“RAS结合域”(RBD)的复合体中只有一个构象，在其“激活区”内的一些突变的构象不受GDP/GTP交换的影响，被发现参与了与另一个RAS结合域--Raf-1的“富哭区(CRD)”的结合。此外，RAS激活区的这些突变被发现削弱了RAS激活Raf-1激酶活性的能力。因此，Raf-1的激活需要Res同时与RBD和CRD结合。此外，Ras 31位的残基(Glu)被Lys取代后，Raf-1的CRD结合活性异常增加，Raf-1的激活能力被抑制。

Several different types of GTP-dependent Ras-binding domains have been identified so far for the GAP/NF1 family, the Raf family, PI3K,RalGDS,and so on, but no apparent sequence homology have been elucidated for them. It is expected that a mutant Ras protein with a binding specificity restricted to a subset of those target proteins is a useful tool for analysis of the complicated Ras signaling pathways. In this study, we made Ha-Ras mutants carrying substitution (s) in the region of residues 21-71, and tested their abilities for association with different Ras-binding domains. Actually, we found several mutants that bind to a restricted range of targets. An NMR analysis showed that some of these mutants do not exhibit the "regional polysterism", which has been observed for the GTP-bound from of the wild-type Ras protein. Therefore, we propose that the "regional polysterism" of Ras allows the binding to various target molecules. In fact, we found that the Ras protein takes only a single conformation in a complex with the "Ras-binding domain (RBD) " of Raf-1 or of RGL.Some mutations of Ras within its "activator region" whose conformations are unaffected by GDP/GTP exchenge, were found to be involved in the binding to another Ras-binding domain, the "Cry-rich domain (CRD), " of Raf-1. Furthermore, these mutations in the activator regions of Ras were found to impair the ability of Ras to activate the Raf-1 kinase activity. Thus, the Raf-1 activation is found to require Res to bind to both RBD and CRD.Furthermore, replacement of the reside (Glu) at position 31 of Ras with Lys increased the Raf-1 CRD binding activity abnormally and inhibited the Raf-1 activation ability of Ras.