Regulation of Cardiac PKA Signaling by cAMP and Oxidants

Abstract

Pathologies, such as cancer, inflammatory and cardiac diseases are commonly associated with long-term increased production and release of reactive oxygen species referred to as oxidative stress. Thereby, protein oxidation conveys protein dysfunction and contributes to disease progression. Importantly, trials to scavenge oxidants by systemic antioxidant therapy failed. This observation supports the notion that oxidants are indispensable physiological signaling molecules that induce oxidative post-translational modifications in target proteins. In cardiac myocytes, the main driver of cardiac contractility is the activation of the β-adrenoceptor-signaling cascade leading to increased cellular cAMP production and activation of its main effector, the cAMP-dependent protein kinase (PKA). PKA-mediated phosphorylation of substrate proteins that are involved in excitation-contraction coupling are responsible for the observed positive inotropic and lusitropic effects. PKA-actions are counteracted by cellular protein phosphatases (PP) that dephosphorylate substrate proteins and thus allow the termination of PKA-signaling. Both, kinase and phosphatase are redox-sensitive and susceptible to oxidation on critical cysteine residues. Thereby, oxidation of the regulatory PKA and PP subunits is considered to regulate subcellular kinase and phosphatase localization, while intradisulfide formation of the catalytic subunits negatively impacts on catalytic activity with direct consequences on substrate (de)phosphorylation and cardiac contractile function. This review article attempts to incorporate the current perception of the functionally relevant regulation of cardiac contractility by classical cAMP-dependent signaling with the contribution of oxidant modification.

Keywords: cardiac myocyte; contractile function; kinase; oxidation; phosphatase.

Figure 1

Scheme summarizing the signaling network between PKA, PP1 and PP2A, their inter-action upon βAR stimulation and cAMP production or direct oxidative modification. PKA phosphorylates and activates inhibitor 1 (I-1), which inhibits PP1. Several B’-PP2A regulatory subunits have been shown to be subject to phosphorylation, B56δ is phosphorylated by PKA, enhancing PP2A activity by dissociation from the A-subunit. PP2A mediates I-1 dephosphorylation. PKA phosphorylates substrate proteins in cardiac myocytes that regulate cardiac contraction, which is counteracted by PP2A and PP1 action. In addition to the regulation by phosphorylation, signaling subunits labeled with a star are susceptible to oxidation. Green: enhanced activity, red: inhibitory effect. The scheme synergizes aspects of the regulation of PKA, PP1 and PP2A activity by phosphorylation and simultaneous oxidation to orchestrate contractility. Numbers 1–9 referring to the respective section in the manuscript.

Figure 2

Structure of PKA-RIα dimerization and docking domain (D/D-domain) in complex with D-AKAP2 [52] (PDB 3IM4). The RIα D/D-domain (blue and dark blue) is defined by an N-terminal helical bundle that promotes regulatory subunit dimerization via hydrophobic interactions. Under oxidative conditions, additional intermolecular disulfide bonds are formed by cysteines at position 17 and 38. The interaction with different AKAPs (grey) is mostly caused by hydrophobic interactions alongside salt bridges. An effect of the interdisulfide bond formation on D/D-domain-AKAP interaction and PKA localization could be shown by different studies [43,48].

Figure 3

Structures of PKA-Cα and PP2A in ribbon representation. (A) cAMP-dependent protein kinase catalytic subunit Table 1. ions [60] (PDB 4WB5). The catalytic subunit of PKA consists of two lobes, the N-lobe consists mainly of β-sheets and the C-lobe comprises α-helical structures. In the latter, a redox-sensitive cysteine, Cys199, is located in the activation loop (yellow) close to the catalytic cleft, which was shown to inhibit kinase activity upon oxidation. Another cysteine susceptible to redox processes, Cys343, is found in the C-terminal tail (C-tail, blue) that spans from the C-lobe to the N-lobe of PKA-Cα. (B) Hetero trimeric holoenzyme of protein phosphatase 2A (PP2A) [61] (PDB 3DW8). The fully active PP2A holoenzyme consists of the core enzyme comprising the scaffold unit PP2A-A (teal) and the enzymatically active subunit PP2A-C (grey) as well as a regulatory subunit, here PP2A-B55 (blue). Localized on the β12-β13 loop of PP2A-C (magenta), there are two redox-sensitive cysteines, Cys266 and Cys269, that contribute to an oxidation-induced inhibition of PP2A-C via disulfide bridge formation.