The N-terminal portion of autoinhibitory element modulates human endothelial nitric-oxide synthase activity through coordinated controls of phosphorylation at Thr495 and Ser1177

• Main Authors: Pei‑Rung Wu, Bo‑Rui Chen, Chi‑Chun Hsieh, Wei‑Chung Lin, Kenneth K. Wu, Yeukuang Hwu, Pei‑Feng Chen
• Format: Article
• Language: English
• Published: Portland Press, Biochemical Society 2014-08-01
• Series: Bioscience Reports
• Subjects: autoinhibitory element; calcium/calmodulin; CaM-binding domain; endothelial nitric-oxide synthase; mobility shift gel; phosphorylation/dephosphorylation
• Online Access: http://www.bioscirep.org/bsr/034/e129/bsr034e129.htm

NO production catalysed by eNOS (endothelial nitric-oxide synthase) 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 AIE (autoinhibitory element) 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 HEK-293 (human embryonic kidney 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 non-stimulated HEK-293 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 with WTeNOS (wild-type eNOS). 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.