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

Abstract

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 nMto 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. Wehave 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. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.