title: The N-terminal portion of autoinhibitory element modulates human endothelial nitric-oxide synthase activity through coordinated controls of phosphorylation at Thr495 and Ser1177.
abstract: 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.
authors.forename: Pei-Rung
authors.initials: PR
authors.lastname: Wu
authors.forename: Bo-Rui
authors.initials: BR
authors.lastname: Chen
authors.forename: Chi-Chun
authors.initials: CC
authors.lastname: Hsieh
authors.forename: Wei-Chung
authors.initials: WC
authors.lastname: Lin
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Wu
authors.affiliation: †Institute of Physics, Academia Sinica, Nankang, Taipei 11529, Taiwan.
authors.forename: Yeukuang
authors.initials: Y
authors.lastname: Hwu
authors.forename: Pei-Feng
authors.initials: PF
authors.lastname: Chen
mesh_terms: D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003470:Culture Media / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation / Q000235:genetics; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics* / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics*
chemical_list: D003470:Culture Media; D009566:Nitrates; D009573:Nitrites; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D002118:Calcium