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

1.通过筛选约50个携带与Raf-1、B-Raf、Ral-GDS、Byr2和酵母腺苷酸环化酶相互作用的单氨基酸取代的H-Ras突变体，我们证实了5种效应蛋白与Ras的相互作用机制存在显著差异，并获得了能够区分效应分子的H-Ras突变体。然而，我们发现在使用这些Ras突变体分析单个效应体的细胞功能时存在一个根本问题。通过荧光偏振法，我们发现，携带单氨基酸取代的Ras效应区对应的合成肽可以不同程度地抑制Ras与不同效应物的相互作用。特异性与全Ras蛋白的特异性无明显区别。我们发现了与富半胱氨酸区（CRR）对应的Raf-1的第二个ras结合位点。Ras激活区的突变使Ras与CRR的结合失效，需要对Ras进行翻译后修饰。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，我们阐明了抗癌基因产物RaplA拮抗Ras功能的机制。Rap1A对CRR具有高亲和力，与Raf-1和Ras形成三重复合物，从而抑制Ras与CRR的结合，从而抑制Ras依赖性的Raf-1激活。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。

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.