| dc.description.abstracteng | PKA is activated by β-adrenergic signaling induced elevation in intracellular cAMP and sequestered into proximity with its substrates by scaffold A-kinase anchoring proteins (AKAPs). PKARIα is a unique isoform of PKA as in response to oxidants it forms two inter-protein disulfide bonds between its regulatory subunits, which directly flank its interaction site with AKAPs. As such, it is probable that the oxidation of PKARIα affects its localization with AKAPs, therefore serving as a regulatory mechanism by which the kinase is targeted to its substrates. This thesis examines the interplay between β-adrenergic and oxidant induced PKARIα regulation and its impact on PKA substrate phosphorylation. In particular, regulation of the mitochondrial fission protein dynamin-related protein 1 (DRP1) is assessed as this is facilitated by the PKARIα scaffold protein Dual-AKAP1 (D-AKAP1).
PKARIα formed a disulfide-dimer during ex vivo Langendorff perfusions with H2O2, which was associated with its translocation to the insoluble fraction of cardiac homogenates. This model of PKARIα oxidation was then replicated in vivo in the context of starvation. In heart, 24 hours starvation increased PKARIα disulfide-dimer formation and PKA-substrate phosphorylation, as detected by a pan-specific “total” PKA substrate antibody, specific phosphorylation of DRP1-S637 was unchanged. In liver, 24 hours starvation increased PKARIα disulfide-dimer formation, which co-fractionated with D-AKAP1. DRP1 also formed higher molecular weight complexes consistent with its phosphorylation by PKA. Identifying whether these changes were mediated by PKARIα phosphorylation of DRP1 was not possible as the antibody failed to produce a specific phospho-signal in immunoblots from liver. Unexpectedly, PKARIα-C17S KI mice showed increased cardiac DRP1-S637 phosphorylation after starvation and also displayed a basal elevations in both PKARIα expression and “total” PKA-substrate phosphorylation
Langendorff perfusion experiments revealed that Na-pyruvate attenuates H2O2 induced cysteine oxidation. Physiologically this was evidenced by abolished responses in left ventricular end diastolic pressure (LVEDP) and coronary flow rate (CFR) in response to H2O2 in the presence of Na-pyruvate. PKARIα disulfide-dimer formation in response to H2O2 was not affected by elevating cAMP with the β-adrenergic agonist isoprenaline. H2O2 attenuated isoprenaline-induced elevations in “total” PKA-substrate phosphorylation which physiologically was reflected by blunted CFR, LVEDP and left ventricular end systolic pressure (LVSP) responsiveness to isoprenaline. However, using the PKARIα-C17S KI mouse these changes were seen to occur independently of PKARIα disulfide-dimer formation.
Taken together, the above findings indicate that PKARIα is modulated by both its oxidation to a disulfide-dimer and cAMP binding. However, the interplay between these two factors remains unclear as evidenced by a failure of cellular models to translate to ex vivo and in vivo scenarios. In the heart, starvation induced disulfide PKARIα does not appear to regulate DRP1. However in the context of liver, promising results indicate that starvation induced disulfide PKARIα may contribute to the protective effects of reduced mitochondrial fission through inhibition of DRP1. | de |