Mechanisms for Non-canonical PKA Signaling Regulation in the Heart
- Author(s): Haushalter, Kristofer Jean
- Advisor(s): Taylor, Susan S
- Patel, Hemal H
- et al.
cAMP-dependent protein kinase (PKAc) is a pivotal cell signaling protein in eukaryotes. The catalytic activity of this enzyme is in part controlled by four functionally non-redundant regulatory subunit proteins of PKA (RIα, RIβ, RIIα and RIIβ), which bind PKAc in tetrameric “holoenzyme” complexes to maintain kinase inactivity until stimulation with cyclic-AMP (cAMP). The allosteric activation mechanism triggered by cAMP binding to R-subunits is well understood. However, unique biochemical features and subcellular localizations of RIα and other PKA proteins in the heart may serve to activate PKAc via non-canonical, cAMP-independent mechanisms. The aim of this work was to examine the role of 1) disulfide bond oxidation at the dimerization/docking (D/D) domain of RIα, 2) localization of PKA proteins at mitochondria in the heart, and 3) phosphorylation of RIα by cGMP-dependent protein kinase (PKG) at the linker region. It was hypothesized that these mechanisms result in enhancement of PKAc activity through decreased regulation by RIα.
First, the effect of cardiac ischemia/reperfusion (I/R) injury on RIα protein expression, oxidation, and regulation of PKAc activity was assessed. Results show that RIα is decreased in expression upon extended time periods of reperfusion injury. Analysis in cardiomyocyte cultures showed that oxidant stress alone is sufficient to modify and down-regulate RIα protein, while simultaneously triggering PKAc activity. Furthermore, overexpression of RIα increases apoptosis in oxidant stressed cells. In correlation with these results, PKA proteins are specifically enriched within subsarcolemmal mitochondria (SSM) of the heart, wherein the expression of RIα is specifically decreased with I/R stress. In our study of RIα phosphorylation, we validated that PKG targets Ser101 in RIα under both in vitro and in cell conditions. Phospho-mimetic mutation of Ser101 (S101E) displayed enhanced PKAc activity without cAMP while maintaining holoenzyme complex formation.
Taken together, these findings support the notion that Type I holoenzymes can be alternatively activated by means other than classical cAMP-mediated processes. Future experiments will be aimed at delineating whether these mechanisms can work synergistically within the context of oxidative stress. The implications of this work translate to a better understanding of the role PKA signaling in heart disease.