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.