Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane
Live-cell imaging of endogenous Ras-GTP illustrates predominant Ras activation at the plasma membrane
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DOI：
10.1038/sj.embor.7400560
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发表时间：
2006-01-01
期刊：
影响因子：
7.7
通讯作者：
Rubio, I
中科院分区：
文献类型：
--
作者：
Augsten, M;Pusch, R;Rubio, I
Ras- gtp成像研究使用Ras效应物c-Raf的Ras结合域（RBD）作为过表达Ras的报告者，对高尔基体中Ras的可能激活产生了不同的结果。我们报道RBD寡聚化为内源性Ras- gtp的可视化提供了探针，避免Ras过表达及其衍生的副作用。RBD寡聚化导致与Ras- gtp的紧密结合和Ras信号通路的中断。与绿色荧光蛋白融合的三聚体RBD探针报告了激动剂诱导的内源性Ras在COS-7、PC12和Jurkat细胞的质膜（PM）上激活，但在高尔基体上没有积累。Ras过表达加剧了PM照明，其对显性阴性RasS17N和药理学操作的敏感性与生物化学评估的Ras- gtp形成一致。我们的数据表明，内源性高尔基定位的Ras不受生长因子的控制，并认为PM是激动剂诱导的Ras激活的主要位点。
Ras-GTP imaging studies using the Ras-binding domain (RBD) of the Ras effector c-Raf as a reporter for overexpressed Ras have produced discrepant results about the possible activation of Ras at the Golgi apparatus. We report that RBD oligomerization provides probes for visualization of endogenous Ras-GTP, obviating Ras overexpression and the side effects derived thereof. RBD oligomerization results in tenacious binding to Ras-GTP and interruption of Ras signalling. Trimeric RBD probes fused to green fluorescent protein report agonist-induced endogenous Ras activation at the plasma membrane (PM) of COS-7, PC12 and Jurkat cells, but do not accumulate at the Golgi. PM illumination is exacerbated by Ras overexpression and its sensitivity to dominant-negative RasS17N and pharmacological manipulations matches Ras-GTP formation assessed biochemically. Our data illustrate that endogenous Golgi-located Ras is not under the control of growth factors and argue for the PM as the predominant site of agonist-induced Ras activation.