Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase
Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase
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DOI：
10.1074/jbc.m005116200
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发表时间：
2000-09-29
影响因子：
4.8
通讯作者：
Michel, T
中科院分区：
文献类型：
--
作者：
Bernier, SG;Haldar, S;Michel, T
内皮细胞中缓激肽 B2 受体的激活会引发一系列由不同信号通路介导的复杂细胞反应，包括刺激丝裂原激活蛋白 (MAP) 激酶级联反应和激活内皮一氧化氮合酶 (eNOS) 亚型。几种蛋白激酶与 eNOS 调节有关，但 MAP 激酶的作用仍不清楚。我们探索了牛主动脉内皮细胞 (BAEC) 中 eNOS 与 MAP 激酶通路成分之间的相互作用，通过免疫共沉淀实验，我们分离出了 eNOS 与 MAP 激酶细胞外信号调节激酶 1 和 2 (ERK1/2) 以及蛋白激酶 Raf-1 和 Akt 的复合物。向 BAEC 中添加缓激肽后几分钟内，eNOS-Raf-1-ERK-Akt 异聚复合物解离，随后在更长时间的激动剂刺激后重新结合。 BAEC 的缓激肽处理导致 ERK 激活，与 eNOS 磷酸化增加相关；体外 ERK 磷酸化 eNOS 显着降低 eNOS 酶活性。 BAEC 中 MAP 激酶直接磷酸化 eNOS 的证据来自于体外使用 [gamma-P-32]ATP 和 ERK 磷酸化从先前用缓激肽或 MAP 激酶抑制剂 PD98059 处理的细胞中分离出的 eNOS 的“反向磷酸化”实验。与未处理的细胞相比，从经缓激肽处理的 BAEC 中分离出的 eNOS 体外 ERK 催化的 P-32 磷酸化显着减弱，表明缓激肽处理导致细胞中 ERK 敏感位点的磷酸化。相反，从用 MAP 激酶抑制剂 PD98059 预处理的内皮细胞中分离出的 eNOS 在体外显示出 ERK 促进的磷酸化增加。综上所述，我们的结果表明，缓激肽诱导的 ERK 激活导致 eNOS 磷酸化和酶抑制，这一过程受到 MAP 激酶途径成员与 eNOS 的可逆关联的影响。
Activation of the bradykinin B2 receptor in endothelial cells initiates a complex array of cellular responses mediated by diverse signaling pathways, including stimulation of the mitogen-activated protein (MAP) kinase cascade and activation of the endothelial isoform of nitric-oxide synthase (eNOS). Several protein kinases have been implicated in eNOS regulation, but the role of MAP kinases remains less well understood. We explored the interactions between eNOS and components of the MAP kinase pathway in bovine aortic endothelial cells (BAEC), Using co-immunoprecipitation experiments, we isolated eNOS in a complex with the MAP kinases extracellular signal-regulated kinases 1 and 2 (ERK1/2) as well as the protein kinases Raf-1 and Akt. Within minutes of adding bradykinin to BAEC, the eNOS-Raf-1-ERK-Akt heteromeric complex dissociated, and it subsequently reassociated following more prolonged agonist stimulation. Bradykinin treatment of BAEC led to the activation of ERK, associated with an increase in phosphorylation of eNOS; phosphorylation of eNOS by ERK in vitro significantly reduced eNOS enzyme activity. Evidence for the direct phosphorylation of eNOS by MAP kinase in BAEC came from "back-phosphorylation" experiments using [gamma-P-32]ATP and ERK in vitro to phosphorylate eNOS isolated from cells previously treated with bradykinin or the MAP kinase inhibitor PD98059. The ERK-catalyzed in vitro P-32 phosphorylation of eNOS isolated from BAEC treated with bradykinin was significantly attenuated compared with untreated cells, indicating that bradykinin treatment led to the phosphorylation of ERK-sensitive sites in cells. Conversely, eNOS isolated from endothelial cells pretreated with the MAP kinase inhibitor PD98059 showed increased ERK-promoted phosphorylation in vitro. Taken together, our results suggest that bradykinin-induced activation of ERK leads to eNOS phosphorylation and enzyme inhibition, a process influenced by the reversible associations of members of the MAP kinase pathway with eNOS.