Shinkai M, Masuda T, Kariya K, Tamada M, Shirouzu M, Yokoyama S, Kataoka T
Department of Physiology II, Kobe University School of Medicine, Japan.
Biochem Biophys Res Commun. 1996 Jun 25;223(3):729-34. doi: 10.1006/bbrc.1996.0964.
Ras is known to possess multiple cellular targets including Raf-1. Here, we measured both direct binding of various H-Ras mutants to two representative mammalian Ras targets, Raf-1 and B-Raf, and the activity of the mutants to stimulate Raf-1 and B-Raf, and analysed the difference in their Ras-interaction mechanisms. B-Raf was shown to share almost the same H-Ras binding-specificity with Raf-1 by examining binding of the H-Ras mutants to Raf-1 and B-Raf in the yeast two-hybrid and in vitro binding assays. Mutants, Y32F, A59E, and V45E bound to Raf-1 in Sf9 cells coexpressing them, but failed to activate Raf-1. On the other hand, Y32F activated B-Raf in a cell-free system which consisted of rat brain cytosol and recombinant MEK. These results suggest that there is a subtle structural difference in requirements for the interaction of Ras with Raf-1 and B-Raf.
已知Ras具有多个细胞靶点，包括Raf-1。在此，我们测定了各种H-Ras突变体与两个代表性的哺乳动物Ras靶点Raf-1和B-Raf的直接结合情况，以及这些突变体刺激Raf-1和B-Raf的活性，并分析了它们在Ras相互作用机制上的差异。通过酵母双杂交和体外结合试验检测H-Ras突变体与Raf-1和B-Raf的结合情况，结果显示B-Raf与Raf-1具有几乎相同的H-Ras结合特异性。Y32F、A59E和V45E突变体在共表达它们的Sf9细胞中与Raf-1结合，但未能激活Raf-1。另一方面，Y32F在由大鼠脑细胞质和重组MEK组成的无细胞系统中激活了B-Raf。这些结果表明，Ras与Raf-1和B-Raf相互作用的需求存在细微的结构差异。
