title: Oxidative stress induced mitochondrial protein kinase A mediates cytochrome c oxidase dysfunction.
abstract: Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.
authors.affiliation: Department of Animal Biology and the Mari Lowe Center for Comparative Oncology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
authors.forename: Satish
authors.initials: S
authors.lastname: Srinivasan
authors.forename: Joseph
authors.initials: J
authors.lastname: Spear
authors.forename: Karunakaran
authors.initials: K
authors.lastname: Chandran
authors.forename: Joy
authors.initials: J
authors.lastname: Joseph
authors.forename: Balaraman
authors.initials: B
authors.lastname: Kalyanaraman
authors.forename: Narayan G
authors.initials: NG
authors.lastname: Avadhani
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D015687:Cell Hypoxia / Q000187:drug effects; D002460:Cell Line; D019069:Cell Respiration / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D051379:Mice; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D009154:Mutation; D017202:Myocardial Ischemia / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D059748:Proteolysis / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D015427:Reperfusion Injury / Q000201:enzymology / Q000378:metabolism / Q000473:pathology
chemical_list: D000975:Antioxidants; D021122:Protein Subunits; D017382:Reactive Oxygen Species; D003576:Electron Transport Complex IV; D017868:Cyclic AMP-Dependent Protein Kinases