1. The N-terminal portion of autoinhibitory element modulates human endothelial nitric-oxide synthase activity through coordinated controls of phosphorylation at Thr495 and Ser1177
- Author
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Pei‑Rung Wu, Bo-Rui Chen, Pei‑Feng Chen, Kenneth K. Wu, Yeukuang Hwu, Wei‑Chung Lin, and Chi‑Chun Hsieh
- Subjects
WTeNOS, wild-type eNOS ,lcsh:Life ,lcsh:QR1-502 ,Biochemistry ,CBD, CaM-binding domain ,DMEM, Dulbecco’s modified Eagle’s medium ,endothelial nitric-oxide synthase ,lcsh:Microbiology ,chemistry.chemical_compound ,Enos ,mobility shift gel ,iNOS, inducible NOS ,Phosphorylation ,HEK-293, human embryonic kidney 293 cell ,Cells, Cultured ,CaM, calmodulin ,ATP synthase ,biology ,phosphorylation/dephosphorylation ,Nitric Oxide Synthase Type III ,EGFP, enhanced green fluorescent protein ,eNOS, endothelial nitric-oxide synthase ,nNOS, neuronal NOS ,GS, goat serum ,calcium/calmodulin ,Biophysics ,chemistry.chemical_element ,S6 ,AIE, autoinhibitory element ,Calcium ,Nitric Oxide ,Nitric oxide ,Dephosphorylation ,autoinhibitory element ,Humans ,Myr−eNOS, myristylation-deficient eNOS ,Molecular Biology ,Nitrites ,Original Paper ,Nitrates ,heNOS, human eNOS ,CaM-binding domain ,H4B, (6R)-5,6,7,8-tetrahydro-L-biopterin ,HEK 293 cells ,Cell Biology ,biology.organism_classification ,Molecular biology ,Culture Media ,lcsh:QH501-531 ,HEK293 Cells ,chemistry ,Mutation ,biology.protein - 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., Our findings demonstrate for the first time that AIE insert exerts its regulatory function by coordinate phosphorylation on eNOS Ser1177 and Thr495, highlighting the importance of AIE in regulating agonist-induced eNOS activation.
- Published
- 2014