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 (O₂ ^.-). Although phosphorylation of eNOS regulates in vivo NO generation, the effects of phosphorylation on eNOS coupling and O ₂^.- generation are unknown. Therefore, we phosphorylated recombinant BH₄-free eNOS in vitro using native kinases and determined O₂^.- generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O₂^.- generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca²⁺ sensitivity of eNOS, such that O₂^.- generation became largely Ca²⁺- independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Cα (PKCα) had no effect on maximum activity or calcium sensitivity but decreased calmodulin binding and increased association with caveolin. In endothelial cells, eNOS-dependent O₂^.- 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 O₂^.- generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O₂^.- 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 O₂^.- generation from the enzyme at low Ca²⁺ concentrations, and PKCα-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.