AMP-activated protein kinase functionally phosphorylates endothelial nitric oxide synthase Ser633.
Endothelial nitric oxide synthase (eNOS) plays a central role in maintaining cardiovascular homeostasis by controlling NO bioavailability. The activity of eNOS in vascular endothelial cells (ECs) largely depends on posttranslational modifications, including phosphorylation. Because the activity of AMP-activated protein kinase (AMPK) in ECs can be increased by multiple cardiovascular events, we studied the phosphorylation of eNOS Ser633 by AMPK and examined its functional relevance in the mouse models. Shear stress, atorvastatin, and adiponectin all increased AMPK Thr172 and eNOS Ser633 phosphorylations, which were abolished if AMPK was pharmacologically inhibited or genetically ablated. The constitutively active form of AMPK or an AMPK agonist caused a sustained Ser633 phosphorylation. Expression of gain-/loss-of-function eNOS mutants revealed that Ser633 phosphorylation is important for NO production. The aorta of AMPKalpha2(-/-) mice showed attenuated atorvastatin-induced eNOS phosphorylation. Nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS) confirmed that eNOS Ser633 was able to compete with Ser1177 or acetyl-coenzyme A carboxylase Ser79 for AMPKalpha phosphorylation. Nano-LC/MS/MS confirmed that eNOS purified from AICAR-treated ECs was phosphorylated at both Ser633 and Ser1177. Our results indicate that AMPK phosphorylation of eNOS Ser633 is a functional signaling event for NO bioavailability in ECs.
Chen
Zhen
Z
Division of Biomedical Sciences, University of California, Riverside, CA 92521, USA.
Peng
I-Chen
IC
Sun
Wei
W
Su
Mei-I
MI
Hsu
Pang-Hung
PH
Fu
Yi
Y
Zhu
Yi
Y
DeFea
Kathryn
K
Pan
Songqin
S
Tsai
Ming-Daw
MD
Shyy
John Y-J
JY
D055372:AMP-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D052242:Adiponectin / Q000378:metabolism; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000069059:Atorvastatin; D002417:Cattle; D002478:Cells, Cultured; D002853:Chromatography, Liquid / Q000379:methods; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D006538:Heptanoic Acids / Q000494:pharmacology; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D036103:Nanotechnology; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D012694:Serine; D015398:Signal Transduction / Q000187:drug effects; D013314:Stress, Mechanical; D053719:Tandem Mass Spectrometry; D013997:Time Factors; D014162:Transfection
D052242:Adiponectin; D006538:Heptanoic Acids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011743:Pyrimidines; D011758:Pyrroles; D034741:RNA, Small Interfering; D012265:Ribonucleotides; C516138:dorsomorphin; D009569:Nitric Oxide; D000620:Aminoimidazole Carboxamide; D012694:Serine; D000069059:Atorvastatin; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; C534978:Prkaa2 protein, mouse; C526181:PRKAA2 protein, human; D055372:AMP-Activated Protein Kinases; C526180:PRKAA1 protein, human; D000103:Acetyl-CoA Carboxylase; C031143:AICA ribonucleotide