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

1.我们分析了Raf-1的富含半胱氨酸的区域（CRR）与Ras的激活子区域之间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（法尼基化）。通过采用荧光偏振法来测量与Ras C-末端对应的合成肽的结合，该合成肽与法呢基基团化学连接，我们表明Ras的法呢基部分是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发挥差异调节活性的分子机制，以及蛋白激酶A对RaplA的磷酸化导致其拮抗Ras功能的活性抑制的分子机制。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的激活.我们阐明了激活酵母腺苷酸环化酶所需的Ras法尼基化的分子机制。我们发现了一种新的相互作用之间的法尼基化的Ras和腺苷酸环化酶相关蛋白CAP的N-末端36-残基区域和腺苷酸环化酶的C-末端之间的复合物，并表明，这第二个相互作用是负责的Ras依赖的腺苷酸环化酶激活法尼基化的刺激作用。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通常可能是Ras激活效应分子所必需的。我们发现了一种新的Ras效应候选PLC 210，它编码一种新形式的磷酸肌醇特异性磷脂酶C，在线虫线虫，也分离了cDNA编码其人类同源物。线虫和人PLC 210都表现出与Ras/RaplA的GTP依赖性结合，并具有磷脂酶C活性。目前正在调查Ras的监管模式。

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.