Hüser M, Luckett J, Chiloeches A, Mercer K, Iwobi M, Giblett S, Sun X M, Brown J, Marais R, Pritchard C
Department of Biochemistry, MRC Toxicology Unit and Division of Biomedical Services, University of Leicester, University Road, Leicester LE1 7RH, UK.
EMBO J. 2001 Apr 17;20(8):1940-51. doi: 10.1093/emboj/20.8.1940.
Raf-1 protein kinase has been identified as an integral component of the Ras/Raf/MEK/ERK signalling pathway in mammals. Activation of Raf-1 is achieved by RAS:GTP binding and other events at the plasma membrane including tyrosine phosphorylation at residues 340/341. We have used gene targeting to generate a 'knockout' of the raf-1 gene in mice as well as a rafFF mutant version of endogenous Raf-1 with Y340FY341F mutations. Raf-1(-/-) mice die in embryogenesis and show vascular defects in the yolk sac and placenta as well as increased apoptosis of embryonic tissues. Cell proliferation is not affected. Raf-1 from cells derived from raf-1(FF/FF) mice has no detectable activity towards MEK in vitro, and yet raf-1(FF/FF) mice survive to adulthood, are fertile and have an apparently normal phenotype. In cells derived from both the raf-1(-/-) and raf-1(FF/FF) mice, ERK activation is normal. These results strongly argue that MEK kinase activity of Raf-1 is not essential for normal mouse development and that Raf-1 plays a key role in preventing apoptosis.
Raf-1蛋白激酶已被确定为哺乳动物Ras/Raf/MEK/ERK信号通路的一个组成部分。Raf-1的激活是通过RAS:GTP结合以及质膜上的其他事件实现的，包括340/341位酪氨酸的磷酸化。我们利用基因靶向技术在小鼠中产生了raf-1基因的“敲除”以及具有Y340F/Y341F突变的内源性Raf-1的rafFF突变体。Raf-1(-/-)小鼠在胚胎发育过程中死亡，在卵黄囊和胎盘中表现出血管缺陷，同时胚胎组织的凋亡增加。细胞增殖不受影响。来自raf-1(FF/FF)小鼠细胞的Raf-1在体外对MEK没有可检测到的活性，然而raf-1(FF/FF)小鼠能存活至成年，可育且具有明显正常的表型。在来自raf-1(-/-)和raf-1(FF/FF)小鼠的细胞中，ERK激活是正常的。这些结果有力地表明，Raf-1的MEK激酶活性对于正常小鼠发育并非必不可少，并且Raf-1在预防细胞凋亡中起关键作用。
