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).
Figure 2.
Inhibition of proteasome 26S resulted in an eNOS monomer accumulation in BAECs.
BAECs were incubated with indicated concentration of lactacystin overnight. The protein was harvested with RIPA buffer and run on SDS-PAGE gel at low temperature (4°C) and the eNOS monomer and dimer content was assessed by western blot and quantified using densitometry. (A) Lactacystin caused a dose-dependent accumulation of eNOS monomer but not dimer; (B) The eNOS monomer and dimer in BAECs were normalized with the controls (0 h) and represented as mean ± SE from four independent experiments. * P<0.05 comparison with control.
Figure 3.
VEGF changed the Serine 1179 phosphorylation status in dimers only and did not alter the phosphorylation status of Threonine 497 in eNOS dimers or monomers.
(A) After serum starvation for two hours, BAECs were treated with VEGF (50 ng/ml) for indicated time. The cell lysates were run on SDS-PAGE gels at low temperature. After transferring the protein to nitrocellulose membranes, they were probed with the indicated antibodies. (B) eNOS dimer Ser1179 phosphorylation density was determined by AlphaImager and normalized with control. The results were representative of three independent experiments. (C) The Thr497 phosphorylation density on eNOS dimer and monomer was determined by AlphaImager and normalized with control. The results were represented from three independent experiments (*P<0.05 comparing to control).
Figure 4.
The PI3K signal pathway is the major pathway causing eNOS Serine 1179 phosphorylation induced by VEGF.
After BAECs were treated with MG132 and Ro318220 or LY294002 overnight followed by VEGF challenge, the cell lysates were harvested for the western blotting. The eNOS Ser1179 and Thr497 phosphorylation density were determined by AlphaImager. The results are representative of three independent experiments. (*P<0.05 by one way ANOVA for monomers relative to untreated control and #P<0.05 by one way ANOVA for dimers relative to untreated control).
Figure 5.
Inhibition of PP2A increased phosphorylation of Serine 1179 in eNOS dimer, but did not alter the eNOS dimer∶monomer ratio in BAECs.
BAECs were treated with lactacystin or okadaic acid overnight and the samples were run on SDS-PAGE gel at low temperature (4°C) followed by western blot to determine eNOS phosphorylation and dimer∶monomer ratio as described in the Materials and Methods.
Figure 6.
Inhibition of Hsp90 resulted in eNOS monomerization without change phosphorylation status of Serine 1179 and Threonine 497 in eNOS dimmer.
(A) BAECs were treated with 20 µM geldanamycin for 48 hours and the samples were run SDS-PAGE gel at low temperature and blotted with the indicated antibodies to determine the changes in phosphorylation status of eNOS and dimers and monomers. The eNOS dimer and monomer densities were determined using AlphaImager. And The Ser1179 and Thr497 phosphorylation density on eNOS dimer were also determined using AlphaImager. All results are representative of three independent experiments. * and # P<0.05 comparison with control. (B) BAECs were silenced with scramble siRNA control (scRNA) or Hsp90 (siHsp90) as indicated concentration for 3 days, the cell lysates were blotted with eNOS or Hsp90 antibody respectively. The eNOS monomer and dimer density were measured using AlphaImager and the average of three independent experiments was presented (* and #P<0.05 compared to eNOS dimer and monomer control respectively).