Elucidation of the Molecular Mechanism Underlying the Stimulatory Effect of Posttranslational Lipid Modification of Ras Protein on Activation of Its Effectors

1.分析了RAF-1富含半胱氨酸区(CRR)与RAS激活区相互作用的分子机制和生理意义，这种相互作用依赖于RAS的翻译后修饰(法尼化)。通过荧光偏振法测定与RAS C-末端对应的合成肽的结合力，我们发现RAS的芳基部分是RAS-Raf相互作用的关键决定因素。我们还阐明了RAS和RaplA对Raf-1和B-Raf的不同调节活性以及蛋白激酶A使Rapla磷酸化导致其拮抗RAS功能的分子机制。基于这些结果，我们提出RAS/RaplA和Raf-CRR之间的相互作用强度必须处于足够的水平才能引起Raf激活。我们阐明了激活酵母腺酰环化酶所需的RAS法尼化的分子机制。我们发现了法尼化的RAS与腺酰环化相关蛋白CAP的N端36位残基和腺酰环化酶的C端之间的一种新的相互作用，并表明这种第二种作用是法尼化对RAS依赖的腺酰环化酶激活的刺激作用。这一结果与Raf的数据一起表明，依赖于法尼化的第二相互作用可能是Ras激活效应分子所必需的。我们在线虫中发现了一个新的RAS效应候选者PLC210，它编码一种新形式的肌醇磷脂酶C，并分离到一个编码其人类同源物的cDNA。人PLC210中的这两种线虫都表现出与RAS/RaplA的GTP依赖结合，并具有磷脂酶C活性。RAS的监管模式目前正在调查中。

1. We analysed the molecular mechanism and the physiological significance of interaction between the cysteine-rich region (CRR) of Raf-1 and the activator region of Ras, which is dependent on posttranslational modification (farnesylation) of Ras. By employing the fluorescence polarization method to measure binding of a synthetic peptide corresponding to the Ras C-terminus, which was chemically attached with famesyl group, we showed that the famesyl moiety of Ras is a critical determinant of the Ras-Raf interaction. We also elucitated the molecular mechanisms by which Ras and RaplA exert differential regulatory activities towards Raf-1 and B-Raf and by which phosphorylation of RaplA by protein kinase A results in inhibition of its activity to antagonize the Ras function. Based on these results, we proposed that the strength of interaction between Ras/RaplA and Raf-CRR must stand on an adequate level to cause Raf activation.2. We elucidated the molecular mechanism by which farnesylation of Ras is required for activation of yeast adenylyl cyclase. We discovered a novel interaction between the farnesylated Ras and a complex between the N-terminal 36-residue region of the 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 with Raf suggested that the farnesylation-dependent second interaction may be generally required for activation of the effector molecules by Ras.3. We discovered a novel Ras-effector candidate PLC210, which encodes a new form of phosphoinositide-specific phospholipase C, in the nematode C.elegans, and also isolated a cDNA encoding its human homologue. Both the nematode amid human PLC210 exhibited GTP-dependent binding to Ras/RaplA and had phospholipase C activity'. The mode of regulation by Ras is now under investigation.