权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Development of a Strategy for Selective Inhibition of a Particular Ras Function by Interfering Ras-Effector Interaction

通过干扰 Ras-效应器相互作用来选择性抑制特定 Ras 功能的策略的开发

基本信息

批准号：
07557333
负责人：
KATAOKA Tohru
金额：
$ 2.05万
依托单位：
Kobe University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

1.By screening about 50 H-Ras mutants carrying single smino acid substitutions for interaction with Raf-1, B-Raf, Ral-GDS,Byr2 and yeast adenylyl cyclase, we demonstrated that significant differences exist in the recognition mechanisms by which the five effector proteins associate with Ras, and obtainedH-Ras mutants that could discriminate the effector molecules. However, we found a fundamental problem in using these Ras mutants to analyze the cellular function of individual effector.2.By employing the fluorescence polarization method, we showed that synthetic peptides corresponding to the Ras effector region carrying single amino acid substitutions could differentially inhibit interaction of Ras with distinct effectors. The specificity was indistinguishable from that with the whole Ras protein.3.We discovered a second Ras-binding site of Raf-1 corresponding to the cysteine-rich region (CRR). Ras-binding ot CRR was abolished by mutations in the activator region of Ras and required posttranslational modification of Ras. Binding of Raf-1 to Ras at both of the Ras-binding sites is required for its activation. Based on this discovery, we elucidated the mechanism by which the anti-oncogene product RaplA antagonizes the Ras function. Rap1A has a high affinity for CRR,forms a triple complex with Raf-1 and Ras, and thereby inhibits the binding of Ras to CRR,resulting in inhibition of Ras-dependent Raf-1 activation.4.We elucidated the molecular mechanism by which posttranlational modification (especially farnesylation) of Ras is required for activation of yeast adenylyl cyclase. Farnesylation of Ras is required for activation of adenylyl cyclase, whereas it has no effect on the binding affinity of cyclase for Ras.The stimulatory effect of farnesylation depends on the association of adenylyl cyclase with the adenylyl cyclase-associated protein CAP.This implies that CAP may be an acceptor for the farnesyl moiety of Ras and mediate the effect of Ras farnesylation.

1.通过对50个H-Ras突变体的筛选，发现它们与Raf-1、B-Raf、Ral-GDS、Byr 2和酵母腺苷酸环化酶的相互作用机制存在显著差异，并获得了能够区分这些效应分子的H-Ras突变体。然而，我们发现利用这些Ras突变体来分析单个效应子的细胞功能存在一个根本性的问题。2.通过荧光偏振法，我们发现Ras效应子区域对应的合成肽带有单个氨基酸取代，可以差异性地抑制Ras与不同效应子的相互作用。发现Raf-1的第二个Ras结合位点，对应于富含半胱氨酸的区域（CRR）。Ras与CRR的结合被Ras激活区的突变所消除，并需要Ras的翻译后修饰。Raf-1在两个Ras结合位点与Ras结合是其激活所必需的。基于这一发现，我们阐明了抑癌基因产物RaplA拮抗Ras功能的机制。Rap 1A对CRR具有很高的亲和力，与Raf-1和Ras形成三重复合物，从而抑制Ras与CRR的结合，从而抑制Ras依赖的Raf-1激活。Ras的法尼基化对腺苷酸环化酶的激活是必需的，但对腺苷酸环化酶与Ras的结合亲和力没有影响，法尼基化的激活作用依赖于腺苷酸环化酶与腺苷酸环化酶相关蛋白CAP的结合，这表明CAP可能是Ras的法尼基部分的受体，并介导Ras法尼基化的作用。