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The Significance of Posttranslational Modification (Farnesylation) of Ras Protein in Activation of Its Effectors

Ras 蛋白翻译后修饰（法呢基化）对其效应子激活的意义

基本信息

批准号：
08457038
负责人：
KATAOKA Tohru
金额：
$ 4.29万
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 (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至无数据
项目状态：
已结题

项目摘要

1. Based on our discovery of the second Ras-binding site of Raf-1 corresponding to the cysteine-rich region (CRR), whose interaction with Ras is abolished by mutations in the activator region of Ras and requires posttranslational modification of Ras, we elucidated the mechanism by which the anti-oncogene product Rap1A antagonizes the Ras function. Rap1A has a high affinity for CRR,forms a ternary complex with Raf-1 and Ras, and thereby inhibits the binding of Ras to CRR,resulting in inhibition of Ras-dependent Raf-1 activation. The binding of Rap1A to Raf-1 CRR also requires posttranslational modification (geranylgeranylation) of RaplA.The antagonistic function of RaplA is determined by the nature of its 31th amino acid residue, which is converted to lysine compared to glutamic acid of Ras.2. We elucidated the molecular mechanism by which posttranslational 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. These results led us to propose a new concept of "isoprenyl group acceptor sites".3. By employing the fluorescence polarization method, we were able to show that a synthetic peptide corresponding to the C-terminus of Ras which was chemically attached with farnesyl group bound specifically to CAP,suggesting that CAP is really an acceptor molecule for the farnesyl moiety of Ras. However, we failed to detect similar interaction of the farnesylated peptide with Raf-1 CRR.This is presumably due to vary low affinity of their interaction.

1.基于我们发现Raf-1的第二个Ras结合位点，对应于富含半胱氨酸的区域（CRR），其与Ras的相互作用被Ras激活区的突变所消除，并且需要Ras的翻译后修饰，我们阐明了抗癌基因产物Rap 1A拮抗Ras功能的机制。Rap 1A对CRR具有高亲和力，与Raf-1和Ras形成三元复合物，从而抑制Ras与CRR的结合，导致Ras依赖性Raf-1活化的抑制。RaplA与Raf-1 CRR的结合还需要RaplA的翻译后修饰（香叶基香叶基化）AaplA的拮抗功能由其第31个氨基酸残基的性质决定，与Ras的谷氨酸相比，其转化为赖氨酸。我们阐明了酵母腺苷酸环化酶激活所需的Ras翻译后修饰（特别是法尼基化）的分子机制。Ras的法尼基化是激活腺苷酸环化酶所必需的，而它对环化酶对Ras的结合亲和力没有影响。法尼基化的刺激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，这意味着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。这些结果使我们提出了“异戊二烯基受体位点”的新概念.通过荧光偏振法，我们发现在Ras的C-末端有一个合成的肽段与CAP特异性结合，表明CAP确实是Ras的法呢基的受体分子。然而，我们没有检测到法尼基化肽与Raf-1 CRR的类似相互作用，这可能是由于它们相互作用的亲和力很低。