Mechanism of Intracellular Signaling via Ras Protein

1.我们发现了一种新的相互作用Ras和它的效应;人Raf和酵母腺苷酸环化酶。这种“第二”相互作用对于Ras完全激活效应子是必不可少的，并且依赖于Ras的“激活剂”区域和翻译后修饰（法尼基化）。第二Ras结合位点位于Raf的富含半胱氨酸的结构域中，并且位于环化酶相关蛋白CAP和腺苷酸环化酶C-末端之间的复合物中。基于这些发现，我们阐明了抑癌基因产物Rap 1拮抗Ras功能的分子机制，Ras和Rap 1对Raf-1和B-Raf发挥不同的调节活性，以及Rap 1被A-激酶磷酸化导致其Ras拮抗活性的抑制。结论：1.第二种相互作用的强度是Ras家族小G蛋白调控效应子的关键因素。通过筛选H-Ras的多个单氨基酸取代突变体与各种Ras效应蛋白的相互作用，我们证明了多种效应蛋白与Ras结合的识别机制存在显着差异，并获得了能够区分效应蛋白的H-Ras突变体。利用这些突变体，我们研究了哪些效应子真正参与了Ras诱导的细胞事件。我们在线虫中发现了一个新的Ras效应子候选PLC-ε，并分离了其人类同源物。人PLC-ε与Ras/Rap 1具有GTP依赖性结合，当与Ras和Rap 1共表达时，PLC-ε分别从细胞质转位到质膜和高尔基体，并具有磷脂酶C活性。因此，PLC-ε是一个真实的Ras效应子。此外，我们还发现了两个新的蛋白质RA-GEF-1和-2，这两个蛋白质都具有与GTP结合的Rap 1直接相关的RA结构域和催化Rap 1上的GDP-GTP交换的GEF结构域。

1. We discovered a novel interaction between Ras and its effectors ; human Raf and yeast adenylyl cyclase. This "second" interaction is essential for full activation of the effectors by Ras, and dependent on the "activator" region and the posttranslational modification (famesylation) of Ras. The second Ras-binding sites are located in the cysteine-rich domain of Raf and in a complex between cyclase-associated protein CAP and the adenylyl cyclase C-terminus. Based on these findings, we elucitated the molecular mechanisms by which the anti-oncogene product Rap1 antagonizes the Ras function, by which Ras and Rap1 exert differential regulatory activities toward Raf-1 and B-Raf, and by which phosphorylation of Rap1 by A-kinase results in inhibition of its Ras-antagonizing activity. We proposed that the strength of the second interaction is a critical determinant of effector regulation by Ras family small G-proteins.2. By screening many single amino acid substitution mutants of H-Ras for interaction with various Ras effector proteins, we demonstrated that significant differences exist in the recognition mechanisms by which the multiple effector proteins associate with Ras, and obtained H-Ras mutants that could discriminate the effectors. Using these mutants, we examined which effector was really involved in some Ras-induced cellular events.3. We discovered a novel Ras-effector candidate PLC-ε in nematode and isolated its human homologue. The human PLC-ε exhibited GTP-dependent binding to Ras/Rap1, and, when coexpressed with Ras and Rap1, was translocated from the cytoplasm to the plasma membrane and the Golgi apparatus, respectively, and had its phospholipase C activity greatly activated. Thus, PLC-ε is a real Ras effector. In addition, we discovered two novel proteins RA-GEF-1 and-2, which possessed both a RA domain associating directly with the GTP-bound Rap1 and a GEF domain catalysing GDP-GTP exchange on Rap1.