Activation of 5'-AMP-activated kinase is mediated through c-Src and phosphoinositide 3-kinase activity during hypoxia-reoxygenation of bovine aortic endothelial cells. Role of peroxynitrite.
AMP-activated kinase (AMPK) is a fuel-sensing enzyme present in most mammalian tissue. In response to a decrease in the energy state of a cell AMPK is phosphorylated and activated by still poorly characterized upstream events. Exposure of bovine aortic endothelial cells (BAEC) to chemically synthesized ONOO- acutely and significantly increased phosphorylation of c-Src, PDK1, AMPK, and its downstream target, acetyl-CoA carboxylase (ACC), without affecting cellular AMP. This novel pathway for AMPK activation was confirmed by the use of pharmacological inhibitors and dominant-negative mutants. Exposure of BAEC to hypoxia-reoxygenation (H/R) caused a biphasic increase in AMPK and ACC phosphorylation, which was prevented by adenoviral overexpression of superoxide dismutase (SOD) or inhibition of nitric-oxide synthase (NOS) implicating a role of ONOO- formed during H/R. Furthermore, dominant-negative mutants of c-Src or kinase-defective PDK1 also blocked H/R-induced AMPK activation indicating that, as with addition of exogenous ONOO-, both c-Src and PI 3-kinase are upstream of AMPK. Moreover, H/R, like ONOO-, significantly increased co-immunoprecipitation of AMPK with c-Src, suggesting that ONOO- favors physical association of AMPK with upstream kinases. Taken together, our results indicate a novel pathway by which H/R via ONOO- activates AMPK in a c-Src-mediated, PI 3-kinase-dependent manner, and suggest that ONOO--induced activation of AMPK might thereby regulate metabolic enzymes, such as ACC.
Zou
Ming-Hui
MH
Vascular Research Laboratory, Department of Surgery, Graduate School of Medicine, The University of Tennessee, Knoxville, Tennessee 37920, USA. mzou@mc.utmck.edu
Hou
Xiu-Yun
XY
Shi
Chao-Mei
CM
Kirkpatick
Stacy
S
Liu
Feng
F
Goldman
Mitchell H
MH
Cohen
Richard A
RA
D064413:3-Phosphoinositide-Dependent Protein Kinases; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase / Q000737:chemistry; D000256:Adenoviridae / Q000378:metabolism; D000263:Adenylate Kinase / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D000860:Hypoxia; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D064413:3-Phosphoinositide-Dependent Protein Kinases; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase; D000263:Adenylate Kinase; D009097:Multienzyme Complexes; D010100:Oxygen; D030421:Peroxynitrous Acid; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D013482:Superoxide Dismutase