S-glutathiolation by peroxynitrite of p21ras at cysteine-118 mediates its direct activation and downstream signaling in endothelial cells
S-glutathiolation by peroxynitrite of p21ras at cysteine-118 mediates its direct activation and downstream signaling in endothelial cells
复制标题
DOI：
10.1096/fj.05-4875fje
复制
发表时间：
2006-01-01
期刊：
影响因子：
4.8
通讯作者：
Cohen, RA
中科院分区：
文献类型：
--
作者：
Clavreul, N;Adachi, T;Cohen, RA
高活性物质过氧亚硝酸盐在病理状态下的内皮细胞中产生，其中超氧阴离子和NO的产生增加。在这里，我们表明，过氧亚硝酸盐添加外源性或内源性产生的响应暴露于NO供体或氧化低密度脂蛋白（oxLDL）增加p21 ras活性在牛主动脉内皮细胞。激活是不依赖于上游元素，而是由于直接靶向p21 ras的可逆的S-谷胱甘肽半胱氨酸硫醇的生物素标记技术证明。p21 ras S-谷胱甘肽过氧亚硝酸盐后的时间过程对应于其Raf-1结合活性和易位到膜的增加。此外，p21 ras S-谷胱甘肽化和激活可以被二硫苏糖醇逆转，证实了二硫键的重要性。S-谷胱甘肽化也促进重组p21 ras的鸟嘌呤核苷酸交换。此外，氧化剂诱导的Mek/Erk和PI 3激酶/Akt的激活被显性阴性和Cys-118 p21 ras突变体废除，后者也阻止了p21 ras的S-谷胱甘肽化。这些结果表明，由NO供体或病理刺激如oxLDL引起的过氧亚硝酸盐触发p21 ras Cys-118的直接S-谷胱甘肽化，这增加了p21 ras活性并介导下游信号传导。
The highly reactive species, peroxynitrite, is produced in endothelial cells in pathological states in which the production of superoxide anion and NO is increased. Here, we show that peroxynitrite added exogenously or generated endogenously in response to exposure to an NO donor or oxidized low-density lipoproteins (oxLDL) increases p21ras activity in bovine aortic endothelial cells. The activation is not dependent on upstream elements but rather is due to direct targeting of p21ras by reversible S-glutathiolation of cysteine thiols as demonstrated by biotin-labeling techniques. The time course of p21ras S-glutathiolation following peroxynitrite corresponds to the increase in its Raf-1 binding activity and translocation to the membrane. Moreover, p21ras S-glutathiolation and activation can be reversed by dithiothreitol, confirming the importance of a disulfide bond. S-glutathiolation also promoted guanine nucleotide exchange of recombinant p21ras. In addition, the oxidant-induced activation of Mek/Erk and PI3 kinase/Akt was abrogated by dominant-negative and Cys-118p21ras mutants, and the latter also prevented S-glutathiolation of p21ras. These results indicate that peroxynitrite arising from NO donors or pathological stimuli such as oxLDL triggers direct S-glutathiolation of p21ras Cys-118, which increases p21ras activity and mediates downstream signaling.