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

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，需要对Ras进行翻译后修饰，我们阐明了抗癌基因产物Rap1A拮抗Ras功能的机制。Rap1A对CRR具有高亲和力，与Raf-1和Ras形成三元配合物，从而抑制Ras与CRR的结合，从而抑制Ras依赖性的Raf-1活化。Rap1A与Raf-1 CRR的结合也需要RaplA的翻译后修饰（geranylgeranyation）。RaplA的拮抗功能是由其第31个氨基酸残基的性质决定的，与ras的谷氨酸相比，它可以转化为赖氨酸。我们阐明了酵母腺苷酸环化酶激活所需的Ras翻译后修饰（特别是法尼化）的分子机制。Ras的法尼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。法尼化的刺激作用取决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的法尼化部分的受体，并介导Ras法尼化的作用。这些结果使我们提出了“异戊烯基受体位点”的新概念。通过荧光偏振法，我们可以发现一个与Ras的c端相对应的合成肽与法尼基基团特异性地结合在CAP上，这表明CAP确实是Ras的法尼基部分的受体分子。然而，我们未能检测到法酰化肽与Raf-1 CRR的类似相互作用。这可能是由于它们相互作用的不同的低亲和力。

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.