Disabling a C-terminal Autoinhibitory Control Element in Endothelial Nitric-oxide Synthase by Phosphorylation Provides a Molecular Explanation for Activation of Vascular NO Synthesis by Diverse Physiological Stimuli
2002; Elsevier BV; Volume: 277; Issue: 21 Linguagem: Inglês
10.1074/jbc.m200258200
ISSN1083-351X
Autores Tópico(s)Hormonal Regulation and Hypertension
ResumoCalmodulin-dependent activation of endothelial nitric-oxide synthase is generally considered to follow a transient increase in intracellular calcium levels. However, a number of physiological stimuli ( e.g. endothelial shear-stress, insulin) are known to activate endothelial nitric oxide (eNOS) via a non-classical, "calcium-independent" pathway. Recent findings demonstrate that such stimuli elicit the phosphorylation of a C-terminal residue in eNOS (Ser 1179 in the bovine isoform), rendering eNOS active at resting levels of intracellular calcium. However, the mechanistic basis for this mode of eNOS activation remains unknown. Protein modeling led us to consider that the C terminus of eNOS may fulfill an autoinhibitory function that can be disrupted by phosphorylation of serine 1179. To test this possibility we contrasted the phenotype of wild type bovine eNOS with that of a mutant lacking C-terminal residues 1179–1205 (CΔ27 eNOS). Despite no observed difference in calmodulin affinity, CΔ27 eNOS exhibited a 5-fold reduction in EC 50 for calcium and a 2–4-fold increase in maximal catalytic activities. In these phenotypic properties, CΔ27 accurately mimics phospho-Ser 1179 wild type eNOS. We conclude that the C terminus imposes a significant barrier to the activation of eNOS by calmodulin binding and that this barrier can be functionally disabled by Ser 1179 phosphorylation-elicited enzyme activation.
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