Mechanism of Intracellular Signaling via Ras Protein

点击翻译按钮获取中文摘要

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.