Abstract 16536: Characterizing the PKA and PKG Phosphoproteomes of the Cardiac Myocyte
Protein phosphorylation is an important post translational modification involved in cellular signaling. Disruption of this balance alters cellular phenotype and in some cases can lead to adverse effects, including those associated with myocardial hypertrophy and heart failure. For example, protein kinase A (PKA) and protein kinase G (PKG) activities have been shown to be altered in animal heart failure models, implying an important role for these kinases. Although activation of PKA acutely enhances cardiac function, chronic stimulation can worsen outcome and increase mortality. Interestingly, protein kinase G (PKG) activity has been shown to be decreased in animal heart failure models and increasing PKG activity improves all aspects of the pathophysiology. Although some proteins and their modifiable amino acids have been identified, the myocardial PKA and PKG-dependent phospho-proteome is not established. In this study state-of-the-art proteomic techniques, based on multiple phosphopeptide enrichment steps and high resolution mass spectrometry of isolated rat cardiac myocyte without and with treatment of 8-bromo-cAMP and 8-bromo-cGMP, were used to defined the PKA and PKG specific phospho-proteome, respectively (4 biological replicates, 2 technical replicates). We identified approximately 1300 phosphorylated residues on 400 proteins, including most of the known PKA and PKG-targeted proteins, some of which had additional novel sites and a host of additional targeted proteins, primarily those localized at the plasma membrane, myofilament/cytoskeletal, or nuclear subproteomes. Interestingly, 33% and 52% of the proteins unique to PKG stimulation were localized in the nucleus and plasma membrane, respectively; 50% and 40% of the proteins unique to PKA stimulation were localized to the nucleus and plasma membrane, respectively. Thus PKA and PKG have a broader effect than anticipated, with unexpected targeting of nuclear transcriptional proteins, indicating that chronic activation or inhibition of these kinases may have long term transcriptional and translational effects, which could possibly contribute to cardiac disease phenotypes.
- © 2012 by American Heart Association, Inc.