Effects of combined phosphorylation at Ser-617 and Ser-1179 in endothelial nitric-oxide synthase on EC50(Ca2+) values for calmodulin binding and enzyme activation.
We have investigated the possible biochemical basis for enhancements in NO production in endothelial cells that have been correlated with agonist- or shear stress-evoked phosphorylation at Ser-1179. We have found that a phosphomimetic substitution at Ser-1179 doubles maximal synthase activity, partially disinhibits cytochrome c reductase activity, and lowers the EC(50)(Ca(2+)) values for calmodulin binding and enzyme activation from the control values of 182 +/- 2 and 422 +/- 22 nm to 116 +/- 2 and 300 +/- 10 nm. These are similar to the effects of a phosphomimetic substitution at Ser-617 (Tran, Q. K., Leonard, J., Black, D. J., and Persechini, A. (2008) Biochemistry 47, 7557-7566). Although combining substitutions at Ser-617 and Ser-1179 has no additional effect on maximal synthase activity, cooperativity between the two substitutions completely disinhibits reductase activity and further reduces the EC(50)(Ca(2+)) values for calmodulin binding and enzyme activation to 77 +/- 2 and 130 +/- 5 nm. We have confirmed that specific Akt-catalyzed phosphorylation of Ser-617 and Ser-1179 and phosphomimetic substitutions at these positions have similar functional effects. Changes in the biochemical properties of eNOS produced by combined phosphorylation at Ser-617 and Ser-1179 are predicted to substantially increase synthase activity in cells at a typical basal free Ca(2+) concentration of 50-100 nm.
Tran
Quang-Kim
QK
Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, USA.
Leonard
Jared
J
Black
D J
DJ
Nadeau
Owen W
OW
Boulatnikov
Igor G
IG
Persechini
Anthony
A
D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D003579:Cytochrome Reductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020125:Mutation, Missense; D052250:Nitric Oxide Synthase Type III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013312:Stress, Physiological / Q000502:physiology
D002147:Calmodulin; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D003579:Cytochrome Reductases; D051057:Proto-Oncogene Proteins c-akt; D002118:Calcium