Published online before print January 8, 2009, doi: 10.1161/CIRCRESAHA.108.187567
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Submitted on May 25, 2008
Revised on December 19, 2008
Accepted on December 30, 2008
AMP-Activated Protein Kinase Functionally Phosphorylates Endothelial Nitric Oxide Synthase Ser633
Zhen Chen ;
From the Division of Biomedical Sciences (Z.C., I.-C.P., W.S., Y.F., K.D., J.Y.-J.S.), Biochemistry and Molecular Biology Graduate Program (I.-C.P.,), and W. M. Keck Proteomics Laboratory (S.P.), Institute for Integrative Genome Biology, University of California, Riverside; Department of Physiology and Pathophysiology (Y.F., Y.Z.), Health Science Center, Peking University, Beijing, China; and Genomics Research Center (M.-I.S., P.-H.H., M.-D.T.) and Institute of Biological Chemistry (M.-D.T.), Academia Sinica, Taipei, Taiwan.
* To whom correspondence should be addressed. E-mail: john.shyy{at}ucr.edu.
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 AMPK
2-/- 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 AMPK 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.
Key words: AMPK • eNOS • endothelial cells • nitric oxide bioavailability • phosphorylation
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