High-density lipoprotein and apolipoprotein AI increase endothelial NO synthase activity by protein association and multisite phosphorylation.
NO propagates a number of antiatherogenic effects in the endothelium, and diminished availability has been associated with vascular disease. Recently it has been reported that phosphorylation of endothelial NO synthase (eNOS) at Ser-1179 is required to increase activity in response to stimuli, including high-density lipoprotein (HDL). The current study was undertaken to further examine the mechanism by which HDL activates eNOS and to specifically determine the role of the major apolipoprotein of HDL, apolipoprotein AI (ApoAI). Phosphorylation of eNOS residues Ser-116, Ser-617, Ser-635, Ser-1179, and Thr-497 after incubation with ApoAI and HDL was examined. There were significant increases in phosphorylation at Ser-116 in response to both HDL and ApoAI and similar magnitudes of dephosphorylation at Thr-497. Ser-1179 phosphorylation increased transiently but returned to basal level after 2.5 min. Data demonstrating activation of AMP activated protein kinase (AMPK) during HDL and ApoAI incubation suggests that AMPK may play a role in activation of eNOS. NO release in response to HDL and ApoAI stimulation in endothelial cells paralleled the time frames of phosphorylation, suggesting a causal relationship. Furthermore, ApoAI was found to associate with eNOS in endothelial cells and bind transfected eNOS in Chinese hamster ovary cells, whereas confocal data demonstrates colocalization of ApoAI and eNOS in the perinuclear region, suggesting a protein-protein interaction. Collectively, the results indicate that HDL and ApoAI increase eNOS activity by multisite phosphorylation changes, involving AMPK activation after protein association between ApoAI and eNOS.
Drew
Brian G
BG
Baker Heart Research Institute, P.O. Box 6492 St. Kilda Road Central, Melbourne, Victoria 8008, Australia.
Fidge
Noel H
NH
Gallon-Beaumier
Gabrielle
G
Kemp
Bruce E
BE
Kingwell
Bronwyn A
BA
D055372:AMP-Activated Protein Kinases; D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism*; D002417:Cattle; D008076:Cholesterol, HDL / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D009097:Multienzyme Complexes / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism
D016632:Apolipoprotein A-I; D008076:Cholesterol, HDL; D009097:Multienzyme Complexes; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases