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.
Highlights
Our results demonstrated that phosphorylation of endothelial NO synthase (eNOS) at Ser-1177 is pivotal in the regulation of O2. generation, altering both the Ca2ϩ sensitivity of the enzyme and maximal rate of product generation, whereas phosphorylation of Thr-495 indirectly affects O2. generation by modulating the binding of proteins known to regulate the activity of the enzyme
Protein Expression, Purification, and Characterization—To mitigate problems with low yield and instability of heNOS when expressed in an E. coli system, recombinant human eNOS was coexpressed with CaM as reported previously [26] and iso
The molecular weight of the purified protein, 135 kDa, is in accordance with the molecular mass for native heNOS monomer [3, 10, 33], and the identity of this band was confirmed by immunoblotting using an anti-eNOS antibody (Fig. 1), and mass spectrometry
Summary
Materials—Akt kinase, PKC␣ kinase, anti-eNOS antibody, anti-phospho-Ser-1177, and anti-phospho-Thr-495 eNOS antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). 2 liters of human eNOS (heNOS)-expressing cells were pelleted and resuspended in 50 ml of lysis buffer containing 40 mM HEPES, pH 7.6, 10% glycerol, 500 mM NaCl, 40 mM imidazole, 1 mM DTT, 10 M BH4, and 5 tablets of protease inhibitor mixture, EDTA free. Protein and Heme Content Determination—Protein concentration of purified heNOS was determined by the Bradford assay from Bio-Rad using a bovine serum albumin standard. Generation by EPR Spin Trapping— Spin-trapping measurements of oxygen radicals were performed in 50 mM Tris-HCl buffer, pH 7.4, containing 0.5 mM NADPH, 0.5 mM Ca2ϩ, 10 g/ml CaM, 15 g/ml purified heNOS, and 25 mM spin trap DEPMPO. Generation from heNOS—A 100-l reaction volume was typically used for EPR measurements, identical to that described above except that Cav-P (400 M final concentration) was added to each reaction. The coverslips with cells were mounted on a glass slide with the antifade mounting medium and viewed with a Zeiss confocal microscope at a magnification of 60ϫ, and images were captured digitally
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
