Modulation by peroxynitrite of Akt- and AMP-activated kinase-dependent Ser1179 phosphorylation of endothelial nitric oxide synthase.
Peroxynitrite (ONOO(-)), a nitric oxide-derived oxidant, uncouples endothelial nitric oxide synthase (eNOS) and increases enzymatic production of superoxide anions (O(2)()) (Zou, M. H., Shi, C., and Cohen, R. A. (2002) J. Clin. Invest. 109, 817-826). Here we studied how ONOO(-) influences eNOS activity. In cultured bovine aortic endothelial cells (BAEC), ONOO(-) increased basal and agonist-stimulated Ser(1179) phosphorylation of eNOS, whereas it decreased nitric oxide production and bioactivity. However, ONOO(-) strongly inhibited the phosphorylation and activity of Akt, which is known to phosphorylate eNOS-Ser(1179). Moreover, expression of an Akt dominant-negative mutant did not prevent ONOO(-)-enhanced eNOS-Ser(1179) phosphorylation. In contrast to Akt, ONOO(-) significantly activated 5'-AMP-activated kinase (AMPK), as evidenced by its increased Thr(172) phosphorylation as well as increased Ser(92) phosphorylation of acetyl-coenzyme A carboxylase, a downstream target of AMPK. Associated with the increased release of O(2)(), ONOO(-) significantly increased the co-immunoprecipitation of eNOS with AMPK. Further, overexpression of the AMPK-constitutive active adenovirus significantly enhanced ONOO(-) up-regulated eNOS-Ser(P)(1179). In contrast, overexpression of a dominant-negative AMPK mutant attenuated the ONOO(-)-enhanced eNOS-Ser(1179) phosphorylation as well as O(2)() release. We conclude that ONOO(-) inhibits Akt and increases AMPK-dependent Ser(1179) phosphorylation of eNOS resulting in enhanced O(2)() release.
Zou
Ming-Hui
MH
Vascular Biology Unit, Whitaker Cardiovascular Institute, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts 02118, USA. mhzou@medicine.bu.edu
Hou
Xiu-Yun
XY
Shi
Chao-Mei
CM
Nagata
Daisuke
D
Walsh
Kenneth
K
Cohen
Richard A
RA
D055372:AMP-Activated Protein Kinases; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000493:pharmacokinetics; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology; D004789:Enzyme Activation; D005799:Genes, Dominant; D009097:Multienzyme Complexes / Q000378:metabolism*; D009154:Mutation; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D015854:Up-Regulation; D015032:Zinc / Q000378:metabolism
D009097:Multienzyme Complexes; D011518:Proto-Oncogene Proteins; D030421:Peroxynitrous Acid; D012694:Serine; D001120:Arginine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D055372:AMP-Activated Protein Kinases; D006152:Cyclic GMP; D015032:Zinc; D010100:Oxygen