Mechanism of Intracellular Signaling via Ras Protein

1. 我们发现Ras和它的效应物之间有一种新的相互作用；人Raf和酵母腺苷酸环化酶。这种“第二次”相互作用对于Ras充分激活效应体至关重要，并且依赖于Ras的“激活子”区域和翻译后修饰（家族化）。第二个ras结合位点位于Raf的富含半胱氨酸的区域，位于环化酶相关蛋白CAP和腺苷酸环化酶c端之间的复合物中。基于这些发现，我们阐明了抗癌基因产物Rap1拮抗Ras功能的分子机制，Ras和Rap1对Raf-1和B-Raf发挥不同的调控活性，以及a激酶磷酸化Rap1抑制其Ras拮抗活性的机制。我们提出，第二次相互作用的强度是Ras家族小g蛋白调控效应的关键决定因素。通过筛选多个与多种Ras效应蛋白相互作用的H-Ras单氨基酸替代突变体，我们发现多种效应蛋白与Ras相互作用的识别机制存在显著差异，获得了能够区分这些效应蛋白的H-Ras突变体。利用这些突变体，我们检查了哪些效应物真正参与了一些ras诱导的细胞事件。我们在线虫中发现了一种新的ras -效应候选物PLC-ε，并分离出了它在人体内的同源物。人PLC-ε与Ras/Rap1表现出gtp依赖性结合，当与Ras和Rap1共表达时，PLC-ε分别从细胞质转移到质膜和高尔基体，其磷脂酶C活性被大大激活。因此，PLC-ε是一个真正的Ras效应。此外，我们还发现了两个新的蛋白RA-GEF-1和2，它们既具有与gtp结合的Rap1直接相关的RA结构域，又具有催化Rap1上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.