Winkler D G, Johnson J C, Cooper J A, Vojtek A B
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
J Biol Chem. 1997 Sep 26;272(39):24402-9. doi: 10.1074/jbc.272.39.24402.
The oncoprotein Ras transforms cells by binding to one or more effector proteins. Effector proteins have been identified by their ability to bind to Ras in the GTP but not GDP form, and by their requirement for the Ras effector domain for binding. The best understood Ras effectors are serine/threonine kinases of the Raf family, but other candidate Ras effectors, including a Ral guanine nucleotide dissociation stimulator and phosphatidylinositol 3-kinase (PI3 kinase) have also been identified. To investigate the mechanism of binding of cRaf-1 to Ras, and to investigate the roles of other candidate Ras effectors in transformation, we have isolated and characterized mutants of activated Ras with decreased binding to cRaf-1 relative to other candidate effectors. Examination of these mutants indicates that surface-exposed residues of Ras outside the minimal effector domain interact differentially with cRaf-1 and other Ras-binding proteins, and that fibroblast transformation correlates with cRaf-1 binding and mitogen-activated protein (MAP) kinase activation. Furthermore, activation of PI3 kinase can occur in the absence of significant MAP kinase activation, suggesting that PI3 kinase activation is a primary effect of Ras.
癌蛋白Ras通过与一种或多种效应蛋白结合来转化细胞。效应蛋白已通过它们与处于GTP而非GDP形式的Ras结合的能力以及它们与Ras效应结构域结合的需求来鉴定。研究得最清楚的Ras效应蛋白是Raf家族的丝氨酸/苏氨酸激酶，但也已鉴定出其他候选Ras效应蛋白，包括Ral鸟嘌呤核苷酸解离刺激因子和磷脂酰肌醇3激酶（PI3激酶）。为了研究cRaf-1与Ras的结合机制，并研究其他候选Ras效应蛋白在转化中的作用，我们分离并鉴定了与其他候选效应蛋白相比与cRaf-1结合减少的活化Ras突变体。对这些突变体的研究表明，最小效应结构域之外的Ras表面暴露残基与cRaf-1和其他Ras结合蛋白的相互作用不同，并且成纤维细胞转化与cRaf-1结合和丝裂原活化蛋白（MAP）激酶激活相关。此外，PI3激酶的激活可以在没有明显MAP激酶激活的情况下发生，这表明PI3激酶激活是Ras的主要效应。
