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, parti...

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Bibliographic Details
Published in:The Journal of biological chemistry 2009-05, Vol.284 (18), p.11892-11899
Main Authors: Tran, Quang-Kim, Leonard, Jared, Black, D.J., Nadeau, Owen W., Boulatnikov, Igor G., Persechini, Anthony
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Animals
Calcium - chemistry
Calcium - metabolism
Calmodulin - chemistry
Calmodulin - genetics
Calmodulin - metabolism
Cattle
Cytochrome Reductases - chemistry
Cytochrome Reductases - genetics
Cytochrome Reductases - metabolism
Enzyme Catalysis and Regulation
Humans
Mutation, Missense
Nitric Oxide Synthase Type III - chemistry
Nitric Oxide Synthase Type III - genetics
Nitric Oxide Synthase Type III - metabolism
Phosphorylation - physiology
Protein Binding - physiology
Proto-Oncogene Proteins c-akt - chemistry
Proto-Oncogene Proteins c-akt - genetics
Proto-Oncogene Proteins c-akt - metabolism
Stress, Physiological - physiology
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Summary: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 EC50(Ca2+) 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 EC50(Ca2+) 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 Ca2+ concentration of 50–100 nm.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M806205200