Fleming I, Fisslthaler B, Dimmeler S, Kemp B E, Busse R
Institut für Kardiovaskuläre Physiologie, Klinikum der J.W.G.-Universität, Frankfurt, Germany.
Circ Res. 2001 Jun 8;88(11):E68-75. doi: 10.1161/hh1101.092677.
The activity of the endothelial nitric oxide synthase (eNOS) can be regulated independently of an increase in Ca(2+) by the phosphorylation of Ser(1177) but results only in a low nitric oxide (NO) output. In the present study, we assessed whether the agonist-induced (Ca(2+)-dependent, high-output) activation of eNOS is associated with changes in the phosphorylation of Thr(495) in the calmodulin (CaM)-binding domain. eNOS Thr(495) was constitutively phosphorylated in porcine aortic endothelial cells and was rapidly dephosphorylated after bradykinin stimulation. In the same cells, bradykinin enhanced the phosphorylation of Ser(1177), which was maximal after 5 minutes, and abolished by the CaM-dependent kinase II (CaMKII) inhibitor KN-93. Bradykinin also enhanced the association of CaMKII with eNOS. Phosphorylation of Thr(495) was attenuated by the protein kinase C (PKC) inhibitor Ro 31-8220 and after PKC downregulation using phorbol 12-myristate 13-acetate. The agonist-induced dephosphorylation of Thr(495) was completely Ca(2+)-dependent and inhibited by the PP1 inhibitor calyculin A. Little CaM was bound to eNOS immunoprecipitated from unstimulated cells, but the agonist-induced dephosphorylation of Thr(495) enhanced the association of CaM. Mutation of Thr(495) to alanine increased CaM binding to eNOS in the absence of cell stimulation, whereas the corresponding Asp(495) mutant bound almost no CaM. Accordingly, NO production by the Ala(495) mutant was more sensitive to Ca(2+)/CaM than the aspartate mutant. These results suggest that the dual phosphorylation of Ser(1177) and Thr(495) determines the activity of eNOS in agonist-stimulated endothelial cells. Moreover, the dephosphorylation of Thr(495) by PP1 precedes the phosphorylation of Ser(1177) by CaMKII. The full text of this article is available at http://www.circresaha.org.
内皮型一氧化氮合酶(eNOS)的活性可通过Ser(1177)的磷酸化独立于Ca(2+)增加进行调节,但仅导致低水平的一氧化氮(NO)生成。在本研究中,我们评估了激动剂诱导的(Ca(2+)依赖性、高产量)eNOS激活是否与钙调蛋白(CaM)结合域中Thr(495)磷酸化的变化相关。eNOS Thr(495)在猪主动脉内皮细胞中组成性磷酸化,缓激肽刺激后迅速去磷酸化。在同一细胞中,缓激肽增强了Ser(1177)的磷酸化,5分钟后达到最大值,并被CaM依赖性激酶II(CaMKII)抑制剂KN-93消除。缓激肽还增强了CaMKII与eNOS的结合。Thr(495)的磷酸化被蛋白激酶C(PKC)抑制剂Ro 31-8220和使用佛波醇12-肉豆蔻酸酯13-乙酸酯下调PKC后减弱。激动剂诱导的Thr(495)去磷酸化完全依赖于Ca(2+),并被PP1抑制剂 calyculin A抑制。未刺激细胞免疫沉淀的eNOS结合的CaM很少,但激动剂诱导的Thr(495)去磷酸化增强了CaM的结合。将Thr(495)突变为丙氨酸在无细胞刺激时增加了CaM与eNOS的结合,而相应的Asp(495)突变体几乎不结合CaM。因此,Ala(495)突变体产生的NO对Ca(2+)/CaM比天冬氨酸突变体更敏感。这些结果表明,Ser(1177)和Thr(495)的双重磷酸化决定了激动剂刺激的内皮细胞中eNOS的活性。此外,PP1介导的Thr(495)去磷酸化先于CaMKII介导的Ser(1177)磷酸化。本文全文可在http://www.circresaha.org获取。