Publication Date:
2014-07-05
Description:
Nitric oxide (·NO) production catalyzed by endothelial nitric-oxide synthase (eNOS) plays an important role in the cardiovascular system. A variety of agonists activate eNOS through the Ser1177 phosphorylation concomitant with Thr495 dephosphorylation, resulting in increased ·NO production with a basal level of calcium. To date, the underlying mechanism remains unclear. We have previously demonstrated that perturbation of the autoinhibitory element (AIE) in the FMN-binding subdomain can also lead to eNOS activation with a basal level of calcium, implying that the AIE might regulate eNOS activation through modulating phosphorylation at Thr495 and Ser1177. Here we generated stable clones in human embryonic kidney 293 (HEK 293) cells with a series of deletion mutants in both the AIE (Δ594–604, Δ605–612, and Δ626–634) and the C-terminal tail (Δ14; deletion of 1164–1177). The expression of Δ594-604 and Δ605-612 mutants in nonstimulated HEK293 cells substantially increased nitrate/nitrite release into the culture medium; the other two mutants, Δ626-634 and Δ1164-1177, displayed no significant difference when compared to WTeNOS. Intriguingly, mutant Δ594-604 showed close correlation between Ser1177 phosphorylation and Thr495 dephosphorylation, and NO production. Our results have indicated that N-terminal portion of AIE (residues 594-604) regulates eNOS activity through coordinated phosphorylation on Ser1177 and Thr495.
Print ISSN:
0144-8463
Electronic ISSN:
1573-4935
Topics:
Biology
,
Chemistry and Pharmacology
