Omerovic J, Hammond D E, Clague M J, Prior I A
The Physiological Laboratory, University of Liverpool, Liverpool, UK.
Oncogene. 2008 Apr 24;27(19):2754-62. doi: 10.1038/sj.onc.1210925. Epub 2007 Nov 12.
The ubiquitously expressed major Ras isoforms: H-, K- and N-Ras, are highly conserved, yet exhibit different biological outputs. We have compared the relative efficiencies with which epidermal or hepatocyte growth factor activates Ras isoforms and the requirement for specific isoforms in the activation of downstream pathways. We find that the relative coupling efficiencies to each Ras isoform are conserved between stimuli. Furthermore, in both cases, inhibition of receptor endocytosis led to reduced N- and H-Ras activation, but K-Ras was unaffected. Acute knockdown of each isoform with siRNA allows endogenous Ras isoform function and abundance to be probed. This revealed that there is significant variation in the contribution of individual isoforms to total Ras across a panel of cancer cell lines although typically K> or =N>>H. Intriguingly, cancer cell lines where a significant fraction of endogenous Ras is oncogenically mutated showed attenuated activation of canonical Ras effector pathways. We profiled the contribution of each Ras isoform to the total Ras pool allowing interpretation of the effect of isoform-specific knockdown on signalling outcomes. In contrast to previous studies indicating preferential coupling of isoforms to Raf and PtdIns-3-kinase pathways, we find that endogenous Ras isoforms show no specific coupling to these major Ras pathways.
普遍表达的主要Ras亚型：H-Ras、K-Ras和N-Ras，具有高度保守性，但表现出不同的生物学效应。我们比较了表皮生长因子或肝细胞生长因子激活Ras亚型的相对效率，以及下游信号通路激活中对特定亚型的需求。我们发现，不同刺激之间与各Ras亚型的相对偶联效率是保守的。此外，在这两种情况下，抑制受体内吞作用会导致N-Ras和H-Ras激活减少，但K-Ras不受影响。用小干扰RNA（siRNA）急性敲低各亚型可探究内源性Ras亚型的功能和丰度。这表明，在一组癌细胞系中，各亚型对总Ras的贡献存在显著差异，尽管通常是K≥N>>H。有趣的是，内源性Ras很大一部分发生致癌突变的癌细胞系显示出经典Ras效应通路的激活减弱。我们分析了各Ras亚型对总Ras库的贡献，从而能够解释亚型特异性敲低对信号转导结果的影响。与之前表明亚型优先偶联至Raf和磷脂酰肌醇-3-激酶信号通路的研究不同，我们发现内源性Ras亚型与这些主要Ras信号通路没有特异性偶联。
