Mineo C, Anderson R G, White M A
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9039, USA.
J Biol Chem. 1997 Apr 18;272(16):10345-8. doi: 10.1074/jbc.272.16.10345.
The transforming activity of artificially membrane-targeted Raf1 suggests that Ras-mediated recruitment of Raf1 to the plasma membrane is an important step in Raf1 activation. Cellular Ras is concentrated in the caveolae, a microdomain of the plasma membrane that is highly enriched in caveolin, glycosylphosphatidylinositol-anchored proteins, and signal transduction molecules. Growth factor stimulation recruits Raf1 to this membrane domain. Whether Ras simply promotes Raf1 association with caveolae membranes or also modulates subsequent activation events is presently unclear. We have identified a ras variant, ras12V,37G, that does not interact with Raf1 but does interact with a mutant raf1, raf1(257L). To examine the role of Ras in the activation of membrane-bound Raf1, raf1CAAX, and raf1(257L)CAAX, membrane-targeted variants of Raf1 and raf1(257L), respectively, were expressed in fibroblasts with or without coexpression of ras12V, 37G. Cell fractionation localized both raf1CAAX and raf1(257L)CAAX to caveolae membranes independent of ras12V,37G expression; however, coexpression of ras12V,37G enhanced the activation of raf(257L)CAAX, but not raf1CAAX, as monitored by induction of cellular transformation, increased Raf kinase activity, and induction of activated MAP kinase. These results suggest that the Ras/Raf1 interaction plays a role in Raf1 activation that is distinct from membrane recruitment.
人工膜靶向Raf1的转化活性表明，Ras介导的Raf1募集到质膜是Raf1激活的重要步骤。细胞内的Ras集中在小窝中，小窝是质膜的一个微区，富含小窝蛋白、糖基磷脂酰肌醇锚定蛋白和信号转导分子。生长因子刺激可将Raf1募集到这个膜结构域。目前尚不清楚Ras是仅仅促进Raf1与小窝膜的结合，还是也调节随后的激活事件。我们鉴定出一种ras变体，ras12V,37G，它不与Raf1相互作用，但与突变型raf1，raf1(257L)相互作用。为了研究Ras在膜结合型Raf1、raf1CAAX和raf1(257L)CAAX激活中的作用，分别在成纤维细胞中表达了Raf1和raf1(257L)的膜靶向变体，同时有或没有共表达ras12V,37G。细胞分级分离将raf1CAAX和raf1(257L)CAAX都定位到小窝膜上，与ras12V,37G的表达无关；然而，通过细胞转化诱导、Raf激酶活性增加和活化的MAP激酶诱导监测发现，共表达ras12V,37G增强了raf(257L)CAAX的激活，但没有增强raf1CAAX的激活。这些结果表明，Ras/Raf1相互作用在Raf1激活中发挥的作用与膜募集不同。
