Endothelial nitric oxide synthase is regulated by tyrosine phosphorylation and interacts with caveolin-1
Endothelial nitric oxide synthase is regulated by tyrosine phosphorylation and interacts with caveolin-1
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DOI：
10.1074/jbc.271.44.27237
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发表时间：
1996-11-01
影响因子：
4.8
通讯作者：
Sessa, WC
中科院分区：
文献类型：
--
作者：
GarciaCardena, G;Fan, R;Sessa, WC
内皮型一氧化氮合酶（eNOS）的磷酸化调控知之甚少。在这里，我们证明，eNOS酪氨酸磷酸化牛主动脉内皮细胞（BAEC）使用P-32代谢标记，然后通过磷酸化氨基酸分析和磷酸化酪氨酸特异性蛋白质印迹。用过氧化氢和蛋白酪氨酸磷酸酶抑制剂原钒酸钠处理BAEC，增加eNOS酪氨酸磷酸化。利用一种新的免疫NOS测定，酪氨酸磷酸化的增加与酶的比活性降低50%有关。由于eNOS定位于质膜小窝中，我们检测了酪氨酸磷酸化的eNOS是否与小窝的外壳蛋白小窝蛋白-1相互作用。来自牛肺微血管内皮细胞的eNOS的免疫沉淀导致小窝蛋白-1的共沉淀。相反，免疫沉淀小窝蛋白-1导致酪氨酸磷酸化eNOS的共沉淀。因此，酪氨酸磷酸化是eNOS的一种新型调节机制，小窝蛋白-1是第一个eNOS相关蛋白。总的来说，这些观察结果提供了一种新的调控机制eNOS和酪氨酸磷酸化可能会影响其活性，亚细胞运输，并与其他小窝蛋白相互作用的蛋白质在小窝。
The regulation of endothelial nitric oxide synthase (eNOS) by phosphorylation is poorly understood. Here, we demonstrate that eNOS is tyrosine-phosphorylated in bovine aortic endothelial cells (BAEC) using P-32 metabolic labeling followed by phosphoamino acid analysis and by phosphotyrosine specific Western blotting. Treatment of BAEC with hydrogen peroxide and the protein tyrosine phosphatase inhibitor, sodium orthovanadate, increases eNOS tyrosine phosphorylation. Utilizing a novel immunoNOS assay, the increase in tyrosine phosphorylation is associated with a 50% decrease in the specific activity of the enzyme. Because eNOS is localized in plasmalemma caveolae, we examined if tyrosine phosphorylated eNOS interacts with caveolin-1, the coat protein of caveolae, Immunoprecipitation of eNOS from bovine lung microvascular endothelial cells resulted in the co-precipitation of caveolin-1. Conversely, immunoprecipitation of caveolin-1 resulted in the co-precipitation of tyrosine-phosphorylated eNOS. Thus, tyrosine phosphorylation is a novel regulatory mechanism for eNOS and caveolin-1 is the first eNOS-associated protein. Collectively, these observations provide a novel regulatory mechanism for eNOS and suggest that tyrosine phosphorylation may influence its activity, subcellular trafficking, and interaction with other caveolin-interacting proteins in caveolae.