Department of Biological Sciences, Universidade Federal de São Paulo/Campus Diadema, São Paulo, Brazil.
Antioxid Redox Signal. 2013 Jan 20;18(3):221-38. doi: 10.1089/ars.2011.4455. Epub 2012 Sep 24.
S-nitrosylation of Cys118 is a redox-based mechanism for Ras activation mediated by nitric oxide (NO) at the plasma membrane.
Ras signaling pathway stimulation by 50 and/or 100 μM of S-nitrosoglutathione (GSNO) causes proliferation of HeLa cells. Proliferation was not observed in HeLa cells overexpressing non-nitrosatable H-Ras(C118S). HeLa cells overexpressing H-Ras(wt) containing the spatiotemporal probe green fluorescent protein (GFP) fused to the Ras-binding domain of Raf-1 (GFP-RBD) incubated with 100 μM GSNO stimulated a rapid and transient redistribution of GFP-RBD to the plasma membrane, followed by a delayed and sustained recruitment to the Golgi. No activation of H-Ras at the plasma membrane occurred in cells overexpressing H-Ras(C118S), contrasting with a robust and sustained activation of the GTPase at the Golgi. Inhibition of Src kinase prevented cell proliferation and activation of H-Ras by GSNO at the Golgi. Human umbilical vein endothelial cells (HUVECs) stimulated with bradykinin to generate NO were used to differentiate cell proliferation and Ras activation at the plasma membrane versus Golgi. In this model, Src kinase was not involved in cell proliferation, whereas Ras activation proceeded only at the plasma membrane, indicating that HUVEC proliferation induced by NO resulted only from stimulation of Ras.
The present work is the first to demonstrate that NO-mediated activation of Ras in different subcellular compartments regulates different downstream signaling pathways.
S-nitrosylation of H-Ras at Cys(118) and the activation of Src kinase are spatiotemporally linked events of the S-nitrosothiol-mediated signaling pathway that occurs at the plasma membrane and at the Golgi. The nonparticipation of Src kinase and the localized production of NO by endothelial NO synthase at the plasma membrane limited NO-mediated Ras activation to the plasma membrane.
Cys118 的 S-亚硝基化是一种基于氧化还原的机制，可介导一氧化氮（NO）在质膜上激活 Ras。
50 和/或 100 μM S-亚硝基谷胱甘肽（GSNO）刺激 Ras 信号通路可引起 HeLa 细胞增殖。在过表达不可硝化的 H-Ras（C118S）的 HeLa 细胞中未观察到增殖。用 100 μM GSNO 孵育的过表达含有 Raf-1 Ras 结合结构域绿色荧光蛋白（GFP）融合蛋白的 H-Ras（wt）的 HeLa 细胞刺激 GFP-RBD 快速而短暂地重新分布到质膜，随后延迟并持续募集到高尔基体。在过表达 H-Ras（C118S）的细胞中，质膜上没有发生 H-Ras 的激活，而在高尔基体上则发生了强大而持续的 GTPase 激活。Src 激酶抑制剂可防止 GSNO 在高尔基体中引起细胞增殖和 H-Ras 的激活。用人脐静脉内皮细胞（HUVEC）用缓激肽刺激产生 NO，以区分质膜与高尔基体中的细胞增殖和 Ras 激活。在该模型中，Src 激酶不参与细胞增殖，而 Ras 激活仅发生在质膜上，表明 NO 诱导的 HUVEC 增殖仅来自 Ras 的刺激。
本研究首次证明，不同亚细胞隔室中 NO 介导的 Ras 激活调节不同的下游信号通路。
H-Ras 在 Cys(118)的 S-亚硝基化和 Src 激酶的激活是 S-亚硝基硫醇介导的信号通路的时空关联事件，该通路发生在质膜和高尔基体上。质膜上内皮型一氧化氮合酶产生的 Src 激酶的非参与和局部 NO 的产生将 NO 介导的 Ras 激活限制在质膜上。
