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

Ras蛋白介导的信号转导机制的结构研究

基本信息

批准号：
06404080
负责人：
YOKOYAMA Shigeyuki
金额：
$ 19.84万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06404080/
关键词：
Ras Signal transdution RalGDS RGL Raf-1 mSosl PC12 cells NMR regional polysterism Ras Cys-rich domain Sos NMR PHドメイン MAPキナーゼ GAP

项目摘要

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.

到目前为止，已经鉴定出GAP/NF1家族、Raf家族、PI3K、RalGDS等几种不同类型的gtp依赖性ras结合结构域，但没有明确的序列同源性。研究人员预计，具有特定结合特异性的Ras突变蛋白将成为分析复杂Ras信号通路的有用工具。在这项研究中，我们制造了在残基21-71区域携带替换的Ha-Ras突变体，并测试了它们与不同ras结合域的结合能力。事实上，我们发现了几种能与有限目标结合的突变体。核磁共振分析表明，这些突变体中的一些不表现出“区域多聚性”，这是在野生型Ras蛋白的gtp结合中观察到的。因此，我们提出Ras的“区域多聚性”允许其与各种靶分子结合。事实上，我们发现Ras蛋白在与Raf-1或RGL的“Ras结合结构域（RBD）”的复合体中只具有单一构象。在其“激活区”内的一些构象不受GDP/GTP交换影响的Ras突变，被发现与Raf-1的另一个Ras结合结构域“Cry-rich domain （CRD）”的结合有关。此外，这些突变在Ras的激活区被发现削弱了Ras激活Raf-1激酶活性的能力。因此，发现Raf-1激活需要Res与RBD和CRD结合。此外，用Lys取代Ras第31位的残基（Glu）异常增加了Raf-1的CRD结合活性，抑制了Ras的Raf-1激活能力。