Endothelial Nitric Oxide Synthase Dimerization Is Regulated by Heat Shock Protein 90 Rather than by Phosphorylation
Figure 1
Dephosphorylation of Serine 1179 and Threonine 497 changed eNOS activity without affecting eNOS structure.
(A) 2 µg purified bovine eNOS was incubated with PP2A for indicated time in the absence or presence of 30 nM okadaic acid. The eNOS sample was run on SDS-PAGE gel at low temperature. Ser1179 and Thr497 phosphorylation status was assessed. The Ser1179 phosphorylation signal in eNOS dimer and monomer was determined by densitometry and represented a bar graph. *P<0.05 by one way ANOVA relative to control for dimers and #P<0.05 for monomers. **P<0.05 for okadaic acid with PP2A treatment vs. PP2A alone for dimers and ## P<0.05 for monomers. (B) 2 µg purified bovine eNOS was incubated with PP1 in the presence or absence of 1 µM PP1 inhibitor 1. The mixtures were run on SDS-PAGE gel at low temperature and blotted with indicated antibodies *P<0.05 by one way ANOVA relative to control for dimers and #P<0.05 for monomers. **P<0.05 PP1 with PP1 inhibitor treatment vs PP1 alone for dimers and ##P<0.05 for monomers. (C) 2 µg purified eNOS was incubated PP2A or PP1 for 2 hours on ice and then Ca2+, CaM and L-arginine, NADPH and MGD-Fe2+ was added and the resulting samples were run EPR. The MGD-Fe2+-NO EPR signal was recorded as described in the material and methods. The graph shows typical eNOS characteristics with PP1 and PP2A inhibitory activity (* and # P<0.01 respectively compared with control by one way ANOVA. n = 4). (D) After PP2A or PP1 treatment, DEPMPO, Ca2+/CaM and NADPH were added to the mixture at indicated concentration and the DEPMPO-OH adduct EPR signal was recorded as described in the material and methods. The bar graph shows typical eNOS superoxide generation capacity with PP1 and PP2A inhibitory effects (* and # P<0.01 compared with control by one way ANOVA with n = 4).