Kennard Simone, Ruan Ling, Buffett Ryan J, Fulton David, Venema Richard C
Vascular Biology Center, Medical College of Georgia, Georgia Regents University, 1459 Laney Walker Blvd., Augusta, Georgia 30912, USA.
Vascular Biology Center, Medical College of Georgia, Georgia Regents University, 1459 Laney Walker Blvd., Augusta, Georgia 30912, USA; Department of Pediatrics, Medical College of Georgia, Georgia Regents University, 1459 Laney Walker Blvd., Augusta, Georgia 30912, USA.
Vascul Pharmacol. 2016 Jun;81:61-8. doi: 10.1016/j.vph.2016.04.003. Epub 2016 Apr 9.
Production of NO by the endothelial nitric oxide synthase (eNOS) has a major role in blood pressure control and suppression of atherosclerosis. In a previous study, we presented evidence implicating the Pin1 prolyl isomerase in negative modulation of eNOS activity in bovine aortic endothelial cells (BAECs). Pin1 recognizes phosphoserine/phosphothreonine-proline motifs in target proteins and catalyzes prolyl isomerization at the peptide bond. In the present study, we show, first, with purified proteins, that Pin1 binds to eNOS directly via the Pin1 WW domain. Binding is enhanced by mimicking phosphorylation of eNOS at S116. Interaction of Pin1 with eNOS markedly reduces eNOS enzymatic activity. Second, in BAECs, we show that TNFα induces ERK 1/2-mediated S116 phosphorylation of eNOS, accompanied by Pin1 binding. TNFα treatment of BAECs results in a reduction in NO release from the cells in a manner that depends on the activities of both Pin1 and ERK 1/2. Evidence is also presented that this mechanism of eNOS regulation cannot occur in rat and mouse cells because there is no proline residue in the mouse and rat amino acid sequences adjacent to the putative phosphorylation site. Moreover, we find that phosphorylation of this site is not detectable in mouse eNOS.
内皮型一氧化氮合酶（eNOS）产生一氧化氮（NO）在血压控制和动脉粥样硬化抑制中起主要作用。在先前的一项研究中，我们提供了证据表明肽基脯氨酰顺反异构酶1（Pin1）在牛主动脉内皮细胞（BAECs）中对eNOS活性起负调节作用。Pin1识别靶蛋白中的磷酸丝氨酸/磷酸苏氨酸-脯氨酸基序，并催化肽键处的脯氨酰异构化。在本研究中，我们首先用纯化的蛋白表明，Pin1通过其WW结构域直接与eNOS结合。通过模拟eNOS在S116位点的磷酸化可增强这种结合。Pin1与eNOS的相互作用显著降低了eNOS的酶活性。其次，在BAECs中，我们表明肿瘤坏死因子α（TNFα）诱导细胞外信号调节激酶1/2（ERK 1/2）介导的eNOS在S116位点的磷酸化，并伴有Pin1结合。TNFα处理BAECs导致细胞中NO释放减少，其方式取决于Pin1和ERK 1/2的活性。我们还提供了证据表明，这种eNOS调节机制在大鼠和小鼠细胞中不会发生，因为在小鼠和大鼠的氨基酸序列中，与假定的磷酸化位点相邻没有脯氨酸残基。此外，我们发现在小鼠eNOS中检测不到该位点的磷酸化。
