S-Nitrosoglutathione and Endothelial Nitric Oxide Synthase-Derived Nitric Oxide Regulate Compartmentalized Ras S-Nitrosylation and Stimulate Cell Proliferation
S-Nitrosoglutathione and Endothelial Nitric Oxide Synthase-Derived Nitric Oxide Regulate Compartmentalized Ras S-Nitrosylation and Stimulate Cell Proliferation
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DOI：
10.1089/ars.2011.4455
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发表时间：
2013-01-01
影响因子：
6.6
通讯作者：
Monteiro, Hugo P.
中科院分区：
文献类型：
--
作者：
Batista, Wagner L.;Ogata, Fernando T.;Monteiro, Hugo P.
目的：Cys 118的S-亚硝基化是一种基于氧化还原的机制，可通过一氧化氮（NO）在质膜上介导Ras活化。结果：50和/或100 μ M的S-亚硝基谷胱甘肽（GSNO）刺激Ras信号通路引起HeLa细胞增殖。在过表达不可亚硝化H-Ras（C118 S）的HeLa细胞中未观察到增殖。HeLa细胞过度表达H-Ras（wt）含有时空探针绿色荧光蛋白（GFP）融合的Ras结合结构域的Raf-1（GFP-RBD）孵育与100 μ M GSNO刺激的GFP-RBD到质膜的快速和短暂的再分布，随后延迟和持续的招聘到高尔基体。H-Ras在质膜上没有激活发生在过表达H-Ras（C118 S）的细胞中，与高尔基体上的GTdR的稳健且持续的激活形成对比。Src激酶的抑制阻止细胞增殖和GSNO在高尔基体激活H-Ras。用缓激肽刺激人脐静脉内皮细胞（HUVECs）产生NO，用于区分细胞增殖和Ras在质膜与高尔基体的激活。在该模型中，Src激酶不参与细胞增殖，而Ras活化仅在质膜上进行，表明NO诱导的HUVEC增殖仅来自Ras的刺激。创新：目前的工作是第一次证明，NO介导的激活Ras在不同的亚细胞区室调节不同的下游信号通路。结论：H-Ras在Cys（118）处的S-亚硝基化和Src激酶的活化是S-亚硝基硫醇介导的信号传导途径的时空关联事件，其发生在质膜和高尔基体处。Src激酶的不参与和内皮NO合酶在质膜上局部产生NO限制了NO介导的Ras对质膜的激活。抗氧化剂。氧化还原信号。18，221-238.
Aims: S-nitrosylation of Cys118 is a redox-based mechanism for Ras activation mediated by nitric oxide (NO) at the plasma membrane. Results: Ras signaling pathway stimulation by 50 and/or 100 mu M 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 mu M 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. Innovation: The present work is the first to demonstrate that NO-mediated activation of Ras in different subcellular compartments regulates different downstream signaling pathways. Conclusion: 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. Antioxid. Redox Signal. 18, 221-238.