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

点击翻译按钮获取中文摘要

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.