Phosphorylation of endothelial nitric-oxide synthase regulates superoxide generation from the enzyme.
In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O(2)* generation are unknown. Therefore, we phosphorylated recombinant BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O(2)* generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O(2)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O(2)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.
Chen
Chun-An
CA
Davis Heart and Lung Research Institute, Columbus, Ohio 43210, USA.
Druhan
Lawrence J
LJ
Varadharaj
Saradhadevi
S
Chen
Yeong-Renn
YR
Zweier
Jay L
JL
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D009569:Nitric Oxide / Q000096:biosynthesis*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism*
D002147:Calmodulin; D013481:Superoxides; D009569:Nitric Oxide; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; C496628:PRKCA protein, human; D051571:Protein Kinase C-alpha; D002118:Calcium