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

Abstract

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 BH(4) is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O(2)*). 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 BH(4)-free eNOS in vitro using native kinases and determined O(2)* generation using EPR spin trapping. Phosphorylation of Ser-1177 by Akt led to an increase (>50%) in maximal O(2)* generation from eNOS. Moreover, Ser-1177 phosphorylation greatly altered the Ca(2+) sensitivity of eNOS, such that O(2)* generation became largely Ca(2+)-independent. In contrast, phosphorylation of eNOS at Thr-495 by protein kinase Calpha (PKCalpha) 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)* 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)* generation occurred. As such, phosphorylation of eNOS at Ser-1177 is pivotal in the direct regulation of O(2)* and NO generation, altering both the Ca(2+) 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)* generation from the enzyme at low Ca(2+) concentrations, and PKCalpha-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals.

FIGURE 1.

Immunoblotting of phosphorylated heNOS at Thr-495 and Ser-1177. Upper panel, a loading control, blotted against eNOS antibody. Middle panel, heNOS was phosphorylated in vitro by PKCα, Akt, or Akt and PKCα at RT for 20 min and blotted against anti-phospho-Thr-495 eNOS polyclonal antibody. Lower panel, heNOS was phosphorylated in vitro by PKCα, Akt, or Akt and PKCα at RT for 20 min and blotted against anti-phospho-Ser-1177 eNOS polyclonal antibody.

FIGURE 2.

EPR spin-trapping of the kinetics of generation from control heNOS and phospho-Ser-1177 heNOS. A, incremental scanning EPR spectra of oxygen free radical generation. The reaction system contains 50 mm Tris-HCl buffer, pH 7.4, 0.5 mm NADPH, 0.5 mm Ca²⁺, 10 μg/ml CaM, 15 μg/ml purified heNOS, and 25 mm spin trap DEPMPO. Spectra were recorded at room temperature with a microwave frequency of 9.863 GHz, 20 milliwatts of microwave power, and 1.0 G modulation amplitude. Center field was 3510 G with 140-G sweep width. Time constant was 328 ms; each spectrum was an 84-s acquisition. B, the continuous time course of generation from control and phospho-Ser-1177 heNOS was measured by fixing the static field at 3501.1 G on the maximum of the 4th peak of the DEPMPO-OOH adduct, as marked by the asterisk. The time constant was 5243 ms. The reaction system and acquisition parameters were otherwise the same as in A.

FIGURE 3.

Effects of phosphorylation on the magnitude of from heNOS. generation rates from control and phosphorylated heNOS were determined from preparations of enzyme by EPR spin-trapping with DEPMPO as described in Fig. 2. The phosphorylation of heNOS by Akt or Akt and PKCα increased its generation by greater than 50%; however, there was no significant effect from the phosphorylation of heNOS by PKCα. Data were expressed as the mean ± S.E., n = 3 (^*, p < 0.001 versus control).

FIGURE 4.

Ca²⁺ dependence of generation from control, phospho-Ser-1177, and phospho-Thr-495 heNOS. generation rates from control, phospho-Ser-1177, and phospho-Thr-495 heNOS were determined by EPR spin-trapping with DEPMPO as described in Fig. 2. In the reaction 100 μm EGTA was included in the assay buffer. The desired Ca²⁺ concentration was achieved by the addition of a concentrated CaCl₂ stock solution. The free [Ca²⁺] was calculated as described under “Experimental Procedures.” generation from eNOS was almost totally blocked by 1 mm l-NAME over the full range of Ca²⁺ concentrations studied. All data points show the relative magnitude of generation compared with the maximal values measured from control heNOS and correspond to the mean ± S.E. from triplicate experiments.

FIGURE 5.

EGTA inactivation of generation by control heNOS and phospho-Ser-1177 heNOS. generation rates from control and phosphoSer-1177 heNOS were determined by EPR spin-trapping with DEPMPO as described in Fig. 2. The effect of phosphorylation of heNOS on the EGTA inactivation of generation was determined. 200 μm Ca²⁺ was included in the reaction. In control experiments, when EGTA was added to a final concentration of 800 μm, there is no measurable generation, although with Akt phosphorylation, even with 1000 μm EGTA, still ∼25% of the generation remained. All data points show the relative magnitude of generation compared with the maximal values measured from control heNOS and correspond to the mean ± S.E. from triplicate experiments. The line fitting of the experimental points for each curve was performed using a sigmoidal function.

FIGURE 6.

CaM dependence of generation from control, phospho-Ser-1177, and phospho-Thr-495 heNOS. generation rate from control, phospho-Ser-1177, and phospho-Thr-495 heNOS was determined from preparations of enzyme by EPR spin-trapping with DEPMPO as described in Fig. 2. The effect of phosphorylation of heNOS on the interaction with CaM under uncoupled (BH₄-free) conditions was determined. In the presence of 0.5 mm CaCl₂, CaM was added to the desired concentration as indicated. The concentration of CaM required for 50% maximal activity of generation (EC₅₀) was determined from CaM dependence of heNOS generation. The EC₅₀ of control heNOS was 208 nm, the EC₅₀ of phospho-Ser-1177 heNOS was 149 nm, and the EC₅₀ of phospho-Thr-495 heNOS was 496 nm. Data were expressed as mean ± S.E., n = 3.

FIGURE 7.

Imaging of generation and immunostaining in BAECs. A, confocal microscopy measurements of generation from uncoupled eNOS in endothelial cells. BH₄ depletion was achieved by incubation of BAECs with 5 mm 2,4-diamino-6-hydroxypyrimidine (DAHP) 18 h at 37 °C. The generation was visualized from BH₄-depleted BAECs loaded with DHE that reacts to form HE, that exhibits red fluorescence. The blue color corresponds to the nuclei stained with DAPI. Measurable fluorescence was seen only when the BH₄-depleted cells were treated with CaI, VEGF, or PMA, and this was inhibited by l-NAME. B, immunostaining using phosphorylation specific anti-eNOS antibodies in BH₄-depleted BAECs. The left column shows the results using the anti-phospho-Ser-1179 antibody. Ser-1179 phosphorylation was detected in BH₄-depleted BAECs when the cells were treated with CaI or VEGF but not with PMA. The right column shows the results using the anti-phospho-Thr-497 antibody, demonstrating that PMA treatment produced strong phosphorylation of Thr-497. These two immunostaining experiments were done in two sets of cells, both under the same conditions.