Coordinated regulation of endothelial nitric oxide synthase activity by phosphorylation and subcellular localization.
Endothelial nitric oxide synthase (eNOS) is regulated by multiple mechanisms including Ca(2+)/calmodulin binding, protein-protein interactions, phosphorylation, and subcellular locations. Emerging evidence suggests that these seemingly independent mechanisms may be closely correlated. In the present study, the interplay between membrane targeting and phosphorylation of eNOS was investigated by using various mutants designed to target specific subcellular locations or to mimic different phospho states. Phospho-mimicking mutations of wild-type eNOS at S635 and S1179 synergistically activated the enzyme. The targeted eNOS mutants to plasma membrane and Golgi complex exhibited higher NO production activities than that of a myristoylation-deficient cytosolic mutant. Phospho-mimicking mutations at S635 and S1179 rescued the activity of the cytosolic mutant and increased those of the plasma membrane- and Golgi-targeted mutants. In contrast, phospho-deficient mutations at these sites led to inactivation of eNOS. Unlike the other targeted mutants, the cytosolic eNOS mutant was unresponsive to cAMP, indicating that membrane association and phosphorylation are required for eNOS activation. These findings suggest that the coordinated interplay between phosphorylation and subcellular localization of eNOS plays an important role in regulating NO production in endothelial cells.
Boo
Yong Chool
YC
Department of Molecular Medicine, Kyungpook National University School of Medicine, 101 Dongindong-2-ga, Junggu, Daegu 700-422, Republic of Korea.
Kim
Hyo Jung
HJ
Song
Hannah
H
Fulton
David
D
Sessa
William
W
Jo
Hanjoong
H
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology; D008969:Molecular Sequence Data; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010766:Phosphorylation
D000242:Cyclic AMP; D052250:Nitric Oxide Synthase Type III; D017868:Cyclic AMP-Dependent Protein Kinases