Phosphorylation of the autoinhibitory insert in endothelial nitric oxide synthase promotes calmodulin binding at low free Ca2+ concentrations

Abstract

The 45‐residue autoinhibitory insert in the endothelial nitric oxide synthase (eNOS) reductase domain inhibits electron transfer, and this effect is counteracted when CaM is bound to the enzyme. We have investigated the effects of phosphorylation at Ser‐617 and Ser‐635 in this region on the Ca2+ sensitivities of CaM binding and CaM‐dependent enzyme activation and on reductase and synthase activities. The mean EC50(Ca2+) values (± SD) for CaM binding to bovine eNOS and mutants with S617D, S635D, S617D/S635D substitutions or lacking the entire autoinhibitory insert were determined to be 220 ± 25, 109 ± 7, 202 ± 21, 117 ± 14, or 47 ± 9 nM, respectively. Corresponding mean EC50(Ca2+) values for eNOS activation were found to be 440 ± 55, 240 ± 28, 461 ± 56, 263 ± 28, or 124 ± 16 nM. The S617D substitution and deletion of the insert were found to increase CaM‐dependent synthase and cytochrome c reductase activities 2‐fold, and to respectively increase CaM‐independent reductase activity 3‐ and 6‐fold. The S635D substitution was found to have no significant effect on any property examined. These results indicate that phosphorylation at Ser‐617 acts synergistically with CaM to counteract the effects of the autoinhibitory insert. Given a resting free Ca2+ concentration in endothelial cells of 50 – 150 nM, phosphorylation at this site also appears to promote binding of CaM to the synthase under basal conditions.Source of research support: NIH Grant GM074887.