Molecular Mechanism of Regulation of Effector Activities by Small GTP-binding Proteins

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1. We showed that the strength of interaction at the Raf cysteine-rich domain (CRD) determines the response of Raf-1 and B-Raf to Ras and Rap1, and that the interaction of Ras/Rap1 with CRD is regulated through phosphorylation of Raf-1 CR2 domain. These results strongly support our hypothesis that the strength of interaction between Ras/Rap1 and Raf-CRD must be in an adequate level to cause Raf activation2. We discovered a novel interaction between famesylated Ras and a complex between adenylyl cyclase-associated protein CAP and the C-terminus of adenylyl cyclase, and showed that this second interaction is responsible for the stimulatory effect of farnesylation on Ras-dependent adenylyl cyclase activation. This result taken together with the data on Raf suggests that the farnesylation-dependent second interaction may be generally required for activation of effector molecules by Ras/Rap1.3. We discovered a novel form of human phosphoinositide-specific phospholipase C, PLCε.PLCε exhibits GTP-dependent binding to both Ras and Rap1. PLCε is translocated from the cytoplasm to the Plasma membrane and the Golgi apparatus when coexpressed with Ras and Rap1, respectively, and its phospholipase C activity is stimulated by Ras. Further, PLCε possesses a stimulatory activity on guanine nucleotide exchange of Rap1 at its CDC25-like domain. These results indicate that PLCε defines a novel category of Ras/Rap1 effector with the ability to amplify its signaling through Rap1 activation.4. We discovered a novel form of guanine nucleotide exchange factor (GEF) for Rap1, RA-GEF.Human RA-GEF-1 binds Rap1-GTP at its RA domain and exhibits GEF activity toward Rap1/2 at its GEF domain. Further, we showd that RA-GEF-1 is translocated to the Golgi apparatus by association with Rap1-GTP and catalyzes activation of Rap1-GDP, thereby causing an amplification of cellular responses induced by Rap1. Similarly, human RA-GEF-2 binds M-Ras-GTP at its RA domain.