The experiments outlined in this proposal investigate the hypothesis that phosphorylation of 20S proteasome core subunits by PKA provides a venue to tune 20S proteolytic activity and heterogeneity of complex formation. Our understanding of proteasome regulation by PKA is critical in elucidating how mechanisms of degradation malfunction during cardiac hypertrophy and heart failure. To date, distinct roles for phosphorylation on proteolytic activity and 20S complex association have not been attributed. We propose three aims: I) To determine functional effects of PKA phosphorylation on murine 20S proteolytic beta subunit activity. Sites of 20S phosphorylation will be pseudo-phosphorylated (Asp) or rendered non-phosphorylatable (Ala) and functional correlates will be established in isolated cardiomyocytes. II) To measure dynamic changes in phosphorylation, subunit association, and PKA translocation to beta 20S subunits during acute beta 1-adrenergic stimulation. Cardiomyocytes will be treated with dobutamine and kinetics of 20S phosphorylation, subunit association, and PKA translocation to the 20S complex will be measured using sophisticated, sensitive mass spectrometric and biochemical methods. Ill) To determine if expression of pseudo-phosphorylated beta5 20S subunits resulting in enhanced proteolytic activity in vivo attenuates the hypertrophic phenotype during pressure overload-induced cardiac hypertrophy. Transgenic mice expressing a tetracycline-repressible, proteolytically-activating 20S mutation will be generated and subjected to transverse aortic constriction (TAC) to induce cardiac hypertrophy. Ventricular function, morphology, histology, 20S associating partners, and a global phosphorylation profile will be serially determined to characterize disease progression and the effects of hyper-activating the 20S core over time. The significance of studies herein lies in unraveling of a novel biological role for the 20S proteasomal catalytic core as an important downstream target of cAMP-dependent regulation, a pathway critical to heart homeostasis in health and disease. The relevance of our findings lies in the discovery of a novel protein degradation pathway to combat hypertrophic cardiomyopathy, thus providing information to guide therapeutic design and application in patients afflicted with this disease.
