Spatiotemporal Regulation of PKA in Response to beta-Adrenergic Stimulation

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DESCRIPTION (provided by applicant): The overall goal of this research is to elucidate the mechanisms involved in regulating cAMP dependent protein kinase (PKA) activity that give rise to high specificity signaling in response to Padrenergic receptor (P-AR) stimulation. Given the broad spectrum of signaling events that are controlled by this kinase, it is crucial that proper phosphorylation by PKA occur at the right time in the right location within a cell, in order to attain selective and specific cellular responses. As such, deregulation of PKA in cardiomyocytes is often associated with heart failure. The tools needed to study live-cell PKA activity have recently become available and will allow for effective exploration of these mechanisms. Specific aim 1 of this project will compare P-AR stimulated spatiotemporal dynamics of PKA activity by stimulating and/or inhibiting p-ARs with receptor subtype specific agonists and antagonists. Specific aim 2 will attempt to determine the roles that membrane microdomains and spatially compartmentalized p-ARs play in the regulation of PKA dynamics by disrupting membrane microdomain structure (via cholesterol depletion) and monitoring local PKA activity. Specific aim 3 will attempt to investigate the roles that A-kinase anchoring proteins (AKAPs) play in regulating P-AR stimulated PKA dynamics by disrupting AKAPs and then monitoring PKA activity at subcellular locations. Each of these aims is to be carried out in cardiomyocytes in an attempt to fill in the current gaps in our knowledge about mechanisms underlying PKA regulation that links receptor activation to specific cardiac output (ie. heart rate, contraction force, relaxation). Additionally, each aim requires live-cell measurement of PKA activity, which will utilize a FRET-based probe for spatiotemporal resolution of PKA activity. This research should provide more in-depth understanding about basic cardiomyocyte signaling mechanisms and aid in the development of treatment and diagnosis of heart failure.