Redox Regulation of Type Iα PKA in the Heart

Abstract

The two regulatory RIα subunits of cAMP-dependent protein kinase (PKA) form interprotein disulfides during oxidative stress which is associated with kinase activation. PKA classical activation involves cAMP binding to the regulatory subunits causing release of catalytic subunits which phosphorylate substrates. Presence of substrate sensitises Type I PKA to cAMP. PKA subcellular localisation is determined by regulatory subunits binding A-kinase anchoring proteins (AKAPs), with RIα oxidation enhancing their binding. I assessed whether thioredoxin (Trx) redox recycles disulfide RIα back to its reduced state. In adult rat ventricular myocytes (ARVMs) pre-treatment with diamide (inducing disulfide) potentiated isoprenaline-induced cardiac Troponin I phosphorylation. Inhibition of monoamine oxidase (MAO), which metabolises monoamines to generate hydrogen peroxide (H2O2), blocked monoamine-induced RIα oxidation. Echocardiography revealed that RIαCys17Ser „redox-dead‟ knock-in (KI) mouse (which I verified cannot form disulfides) hearts had larger left ventricles during diastole and systole and demonstrated systolic dysfunction. Cytosolic RIα disulfide translocated to membrane and myofilament in isolated WT hearts in response to oxidant. Dual-specificity AKAP (D-AKAP) 1, D-AKAP2, AKAP220 and α/β tubulin were not co-purified with WT or KI RIα after cAMP-affinity capture. LC-MS/MS showed that the PKA substrates, alpha-enolase and ChChd3 did co-purify, but the AKAPs associated with these (myomegalin and sphingosine kinase interacting protein, respectively) were not co-captured. Trx recycled disulfide RIα back to its reduced state shown by an RIα-dependent decrease in NADPH fluorescence and RIα disulfide accumulation in ARVMs treated with Trx reductase inhibitors. In conclusion, whilst monoamines classically elevate cAMP to activate PKA, their degradation by MAO generates H2O2 which induces disulfide formation. Disulfide RI translocates from cytosol to membrane and myofilament in response to oxidant, but whether this is due to an increased affinity for AKAPs remains unclear. Disulfide-induced sensitisation of PKA to cAMP is consistent with the idea it increases RIα affinity for AKAPs, so targeting PKA close to substrate to enable substrate-induced activation

Date of Award	1 Jul 2013
Original language	English
Awarding Institution	King's College London
Supervisor	Philip Eaton (Supervisor) & Michael Marber (Supervisor)