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

1.我们发现Raf富含半胱氨酸结构域（CRD）的相互作用强度决定了Raf-1和B-Raf对Ras和Rap 1的反应，并且Ras/Rap 1与CRD的相互作用通过Raf-1 CR2结构域的磷酸化来调节。这些结果强烈支持我们的假设，Ras/Rap 1和Raf-CRD之间的相互作用的强度必须在一个足够的水平，导致Raf激活2。我们发现了一种新的相互作用之间的法呢基化的Ras和腺苷酸环化酶相关蛋白CAP和腺苷酸环化酶的C-末端之间的复合物，并表明，这第二个相互作用是负责的刺激作用的Ras依赖的腺苷酸环化酶的激活。这一结果与Raf的数据一起表明，法尼基化依赖的第二次相互作用可能是Ras/Rap1.3激活效应分子所必需的。我们发现了一种新的人磷脂酰肌醇特异性磷脂酶C（PLCε），PLCε与Ras和Rap 1均具有GTP依赖性结合。当PLCε分别与Ras和Rap 1共表达时，PLCε从细胞质转移到质膜和高尔基体，并且其磷脂酶C活性被Ras激活。此外，PLCε对Rap 1在其CDC 25样结构域的鸟嘌呤核苷酸交换具有刺激活性。这些结果表明PLCε定义了一类新的Ras/Rap 1效应子，具有通过Rap 1激活放大其信号传导的能力.我们发现了Rap 1的一种新的鸟嘌呤核苷酸交换因子（GEF），RA-GEF-1在其RA结构域与Rap 1-GTP结合，在其GEF结构域对Rap 1/2具有GEF活性。此外，我们发现RA-GEF-1通过与Rap 1-GTP结合而易位到高尔基体，并催化Rap 1-GDP的活化，从而引起Rap 1诱导的细胞反应的放大。类似地，人RA-GEF-2在其RA结构域结合M-Ras-GTP。

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.