Phosphorylation of endothelial nitric-oxide synthase regulates superoxide generation from the enzyme

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH4 is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O-2(center dot) over bar). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O-2(center dot) over bar generation are unknown. Therefore, we phosphorylated recombinant BH4- free eNOS in vitro using native kinases and determined O2 (center dot) over bar generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (> 50%) in maximal O-2(center dot) over bar generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca2+ sensitivity of eNOS, such that O-2(center dot) over bar generation became largely Ca2+-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase C alpha(PKC alpha) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O-2(center dot) over bar generation was stimulated by vascular endothelial growth factor that induced phosphorylation of Ser-1177. With PKC activation that led to phosphorylation of Thr-495, no inhibition of O-2(center dot) over bar generation occurred. As such, phosphorylation of eNOS at Ser- 1177 is pivotal in the direct regulation of O-2(center dot) over bar and NO generation, altering both the Ca2+ sensitivity of the enzyme and rate of product formation, whereas phosphorylation of Thr-495 indirectly affects this process through regulation of the calmodulin and caveolin interaction. Thus, Akt-mediated phosphorylation modulates eNOS uncoupling and greatly increases O-2(center dot) over bar generation from the enzyme at low Ca2+ concentrations, and PKC alpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.