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.
Calmodulin-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 (CDelta27 eNOS). Despite no observed difference in calmodulin affinity, CDelta27 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, CDelta27 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.
Lane
Paul
P
Department of Pharmacology and the Program in Biochemistry and Structural Biology, Weill Medical College of Cornell University, New York, New York 10021, USA.
Gross
Steven S
SS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D017931:DNA Primers; D011994:Recombinant Proteins; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D002118:Calcium