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。基于我们发现与富含半胱氨酸的区域（CRR）相对应的RAF-1的第二个RAS结合位点，其与RAS的相互作用被RAS激活剂区域的突变所取消，并需要RAS的RAS翻译后修饰，我们阐明了RAP1A RAP1A RAPADARY的机制。 RAP1A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，从而抑制Ras依赖性RAF-1激活。 RAP1A与RAF-1 CRR的结合还需要RAPLA的翻译后修饰（香烷基苯基化）。RAPLA的拮抗功能取决于其31th氨基酸残基的性质，该氨基酸残基的性质与Ras.2相比，该氨基酸残基的性质转化为赖氨酸。2。我们阐明了酵母腺苷环酶激活RAS需要翻译后修饰（尤其是Farneylation）的分子机制。 Ras的Farnesylation是激活腺苷酸环化酶所必需的，而它对Cyclase对Ras的结合亲和力没有影响。法尼化的刺激作用取决于腺苷酸环化酶与腺苷酸环酶相关的蛋白质帽的关联。这意味着CAP可能是RAS Farnesyl部分的受体，并介导Ras Farnesylation的效果。这些结果使我们提出了一个新的概念，即“异丙基团受体站点” .3。通过采用荧光极化方法，我们能够证明与Ras的C末端相对应的合成肽，该肽与CAP专门结合的Farnesyl基团化学连接，这表明CAP确实是RAS Farnesyl ras部分的受体分子。但是，我们未能检测到法尼肽与RAF-1 CRR的相似相互作用。这可能是由于其相互作用的低亲和力变化。