Kishida S, Koyama S, Matsubara K, Kishida M, Matsuura Y, Kikuchi A
Department of Biochemistry, Hiroshima University School of Medicine, Japan.
Oncogene. 1997 Dec 11;15(24):2899-907. doi: 10.1038/sj.onc.1201473.
Ral GDP dissociation stimulator (RalGDS), a putative effector protein of Ras, stimulated the GDP/GTP exchange reaction of the post-tanslationally lipid-modified but not the unmodified form of Ral in response to epidermal growth factor in COS cells. The RalGDS action on Ral was enhanced by an active form of Ras but not a Ras mutant which was not post-translationally modified in the cells. The RalGDS activity was inhibited by acidic membrane phospholipids such as phosphatidylinositol and phosphatidylserine but not by phosphatidylcholine or phosphatidylethanolamine in vitro. The post-translationally modified form but not unmodified form of Ras, Ral, and Rap were incorporated in liposomes consisting of these phospholipids. When Ral was incorporated alone in the liposomes, RalGDS did not stimulate the dissociation of GDP from Ral. When Ral was incorporated with the GTP-bound form of Ras in the liposomes, RalGDS stimulated the dissociation of GDP from Ral, while the GDP-bound form of Ras did not affect the RalGDS action. The Ras-dependent Ral activation through RalGDS required the Ras-binding domain of RalGDS. Rap, which shared the same effector loop as Ras, also stimulated the dissociation of GDP from Ral through RalGDS in the liposomes, although Rap did not enhance the RalGDS action in COS cells. Taken together with our previous observations that Ras recruits RalGDS to the membrane, these results indicate that the post-translational modifications of Ras and Ral are important for Ras-dependent Ral activation through RalGDS and that colocalization of Ras and Ral on the membrane is necessary for Ral activation in intact cells.
Ral GDP解离刺激因子（RalGDS）是一种假定的Ras效应蛋白，在COS细胞中，响应表皮生长因子刺激，它能促进翻译后经脂质修饰而非未修饰形式的Ral的GDP / GTP交换反应。Ras的活性形式可增强RalGDS对Ral的作用，但细胞中未经翻译后修饰的Ras突变体则无此作用。体外实验中，RalGDS活性受酸性膜磷脂如磷脂酰肌醇和磷脂酰丝氨酸抑制，但不受磷脂酰胆碱或磷脂酰乙醇胺抑制。翻译后修饰形式而非未修饰形式的Ras、Ral和Rap可整合到由这些磷脂组成的脂质体中。当Ral单独整合到脂质体中时，RalGDS不会刺激Ral上GDP的解离。当Ral与GTP结合形式的Ras一起整合到脂质体中时，RalGDS会刺激Ral上GDP的解离，而GDP结合形式的Ras则不影响RalGDS的作用。通过RalGDS的Ras依赖性Ral激活需要RalGDS的Ras结合结构域。与Ras共享相同效应环的Rap，在脂质体中也可通过RalGDS刺激Ral上GDP的解离，尽管Rap在COS细胞中不会增强RalGDS的作用。结合我们之前关于Ras将RalGDS募集到膜上的观察结果，这些结果表明，Ras和Ral的翻译后修饰对于通过RalGDS的Ras依赖性Ral激活很重要，并且在完整细胞中，Ras和Ral在膜上的共定位对于Ral激活是必要的。
