Marais R, Light Y, Paterson H F, Marshall C J
CRC Centre for Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, London, UK.
EMBO J. 1995 Jul 3;14(13):3136-45. doi: 10.1002/j.1460-2075.1995.tb07316.x.
A central feature of signal transduction downstream of both receptor and oncogenic tyrosine kinases is the Ras-dependent activation of a protein kinase cascade consisting of Raf-1, Mek (MAP kinase kinase) and ERKs (MAP kinases). To study the role of tyrosine kinase activity in the activation of Raf-1, we have examined the properties of p74Raf-1 and oncogenic Src that are necessary for activation of p74Raf-1. We show that in mammalian cells activation of p74Raf-1 by oncogenic Src requires pp60Src to be myristoylated and the ability of p74Raf-1 to interact with p21Ras-GTP. The Ras/Raf interaction is required for p21Ras-GTP to bring p74Raf-1 to the plasma membrane for phosphorylation at tyrosine 340 or 341, probably by membrane-bound pp60Src. When oncogenic Src is expressed with Raf-1, p74Raf-1 is activated 5-fold; however, when co-expressed with oncogenic Ras and Src, Raf-1 is activated 25-fold and this is associated with a further 3-fold increase in tyrosine phosphorylation. Thus, p21Ras-GTP is the limiting component in bringing p74Raf-1 to the plasma membrane for tyrosine phosphorylation. Using mutants of Raf-1 at Tyr340/341, we show that in addition to tyrosine phosphorylation at these sites, there is an additional activation step resulting from p21Ras-GTP recruiting p74Raf-1 to the plasma membrane. Thus, the role of Ras in Raf-1 activation is to bring p74Raf-1 to the plasma membrane for at least two different activation steps.
受体酪氨酸激酶和致癌酪氨酸激酶下游信号转导的一个核心特征是由Raf-1、Mek（丝裂原活化蛋白激酶激酶）和ERK（丝裂原活化蛋白激酶）组成的蛋白激酶级联反应的Ras依赖性激活。为了研究酪氨酸激酶活性在Raf-1激活中的作用，我们检测了激活p74Raf-1所必需的p74Raf-1和致癌性Src的特性。我们发现，在哺乳动物细胞中，致癌性Src激活p74Raf-1需要pp60Src进行肉豆蔻酰化以及p74Raf-1与p21Ras-GTP相互作用的能力。p21Ras-GTP需要Ras/Raf相互作用才能将p74Raf-1带到质膜上，可能由膜结合的pp60Src在酪氨酸340或341处进行磷酸化。当致癌性Src与Raf-1共表达时，p74Raf-1被激活5倍；然而，当与致癌性Ras和Src共表达时，Raf-1被激活25倍，这与酪氨酸磷酸化进一步增加3倍相关。因此，p21Ras-GTP是将p74Raf-1带到质膜进行酪氨酸磷酸化的限制因素。使用Tyr340/341处的Raf-1突变体，我们发现除了这些位点的酪氨酸磷酸化外，p21Ras-GTP将p74Raf-1募集到质膜还会导致一个额外的激活步骤。因此，Ras在Raf-1激活中的作用是将p74Raf-1带到质膜进行至少两个不同的激活步骤。
