Effects of phosphorylation at Tyr‐659 in endothelial nitric oxide synthase on its interaction with calmodulin and catalytic activity

eNOS phosphorylation at Tyr‐657 (659 in the bovine sequence) was recently shown to inhibit NO production in cells. Here we show the in vitro effects of Tyr‐659 phosphorylation on eNOS activity and interaction with calmodulin (CaM). Phosphomimetic substitution (Asp) at Tyr‐659 abolishes eNOS cytochrome C reductase activity and NADPH oxidase activity. Adding single or combined Asp substitutions at Ser‐617, Ser‐635, and Ser‐1179 does not change this effect. Single Asp mutation at Tyr‐659 substantially increases the Ca 2+ sensitivity for eNOS‐CaM binding, lowering the EC50(Ca 2+ ) for this interaction from 180 ± 2 nM to 45 ± 1.4 nM (p<0.001). Adding Asp substitutions at Ser‐617, Ser‐635, and Ser‐1179 attenuates this effects, increasing EC50(Ca 2+ ) values to ~ 80 nM (p<0.001). Treatment of either wild‐type or S617/1179DeNOS with purified proline‐rich tyrosine kinase (PYK‐2) abolishes eNOS activity, which is restored by ~ 70% by treatment of PYK2‐pretreated eNOS with tyrosine phosphatase. The data suggest that phosphorylation at Tyr‐659 abolishes eNOS activity yet turns the synthase into a very sensitive CaM‐binding protein that can bind CaM at very low basal Ca 2+ concentration in cells.