Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms -: Role of protein kinase A
Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms -: Role of protein kinase A
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DOI：
10.1074/jbc.m108789200
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发表时间：
2002-02-01
影响因子：
4.8
通讯作者：
Jo, HJ
中科院分区：
文献类型：
--
作者：
Boo, YC;Sorescu, G;Jo, HJ
最近，我们已经表明剪切应力通过牛主动脉内皮细胞（BAEC）中的蛋白激酶B/Akt（Akt）依赖性机制刺激NO产生（Go，Y. M.，布，Y。C.的方法，帕克，H.，Maland，M. C.的方法，帕特尔河，普里查德，K。一、小的，Fujio，Y.，沃尔什，K.，Darley-Usmar，V.，和Jo，H.（2001）J.Appl.Physiol.91，15741581）。Akt被认为通过直接磷酸化内皮一氧化氮合酶（eNOS）的Ser（1179）残基（eNOS-S-1179）来调节NO的剪切依赖性产生，但尚未报道使用特异性抑制剂或显性失活突变体（Akt（AA）或Akt（AAA））的关键评价。此外，其他激酶，包括蛋白激酶A（PKA）和AMP激酶也显示磷酸化eNOS-S-1179。在这里，我们表明，剪切依赖性的eNOSS 1179磷酸化介导的Akt的独立，但PKA依赖，机制。如果细胞被血管内皮生长因子（VEGF）刺激，则通过使用重组腺病毒构建体在BAEC中表达Akt（AA）或Akt（AAA）抑制eNOS-S-1179的磷酸化，但不受剪切应力的刺激。如前所述，Akt（AA）的表达抑制剪切依赖性NO产生，表明Akt仍然是NO产生的重要调节因子。进一步的研究表明，PKA的选择性抑制剂H89可抑制eNOS-S-1179的剪切依赖性磷酸化和NO的产生。相反，H89不抑制通过在BAEC中表达组成型活性Akt突变体（Akt（Myr））诱导的eNOS-S' 179的磷酸化，表明抑制剂不影响Akt途径。单独的S-溴-cAMP以H89敏感的方式在5分钟内磷酸化eNOS-S-1179而不激活Akt。总的来说，这些结果表明，剪切刺激磷酸化的eNOS-S-1179在PKA依赖性，但Akt的非依赖性的方式，而NO的生产是由依赖于PKA和Akt的机制调节。Akt和PKA之间的协调相互作用可能是eNOS活性响应于生理刺激（如剪切应力）而调节的重要机制。
Recently, we have shown that shear stress stimulates NO. production by the protein kinase B/Akt (Akt)-dependent mechanisms in bovine aortic endothelial cells (BAEC) (Go, Y. M., Boo, Y. C., Park, H., Maland, M. C., Patel, R., Pritchard, K. A., Jr., Fujio, Y., Walsh, K., Darley-Usmar, V., and Jo, H. (2001) J. Appl. Physiol. 91,15741581). Akt has been believed to regulate shear-dependent production of NO. by directly phosphorylating endothelial nitric-oxide synthase (eNOS) at the Ser(1179) residue (eNOS-S-1179), but a critical evaluation using specific inhibitors or dominant negative mutants (Akt(AA) or Akt(AAA)) has not been reported. In addition, other kinases, including protein kinase A (PKA) and AMP kinase have also shown to phosphorylate eNOS-S-1179. Here, we show that shear-dependent phosphorylation of eNOSS1179 is mediated by an Akt-independent, but a PKA-dependent, mechanism. Expression of Akt(AA) or Akt(AAA) in BAEC by using recombinant adenoviral constructs inhibited phosphorylation of eNOS-S-1179 if cells were stimulated by vascular endothelial growth factor (VEGF), but not by shear stress. As shown before, expression of Akt(AA) inhibited shear-dependent NO. production, suggesting that Akt is still an important regulator in NO production. Further studies showed that a selective inhibitor of PKA, H89, inhibited shear-dependent phosphorylation of eNOS-S-1179 and NO. production. In contrast, H89 did not inhibit phosphorylation of eNOS-S' 179 induced by expressing a constitutively active Akt mutant (Akt(Myr) in BAEC, showing that the inhibitor did not affect the Akt pathway. S-Bromo-cAMP alone phosphorylated eNOS-S-1179 within 5 min without activating Akt, in an H89-sensitive manner. Collectively, these results demonstrate that shear stimulates phosphorylation of eNOS-S-1179 in a PKA-dependent, but Akt-independent manner, whereas the NO. production is regulated by the mechanisms dependent on both PKA and Akt. A coordinated interaction between Akt and PKA may be an important mechanism by which eNOS activity is regulated in response to physiological stimuli such as shear stress.