AKAP Integration of Phosphorylation and Ubiquitin Signaling

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PROJECT SUMMARY Every heartbeat involves the coordination of local cAMP and Ca2+ signaling events that elicit the contraction of cardiomyocytes. This recurrent process necessitates the reuptake of cytoplasmic Ca2+ through the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2). Pathophysiological changes in SERCA2 protein expression following myocardial infarction have been linked to ubiquitin-mediated proteasomal degradation of the channel. Thus, molecular mechanisms that remove ubiquitin from SERCA2 have the potential to be cardioprotective. The Scott lab have discovered a macromolecular complex containing enzymes of the protein phosphorylation and protein ubiquitination machinery. This new signaling unit may modulate SERCA2 levels at the sarcoplasmic reticulum to facilitate healthy cardiomyocyte function. A more detailed investigation of these molecular and cellular findings is mandated by evidence that cardiovascular diseases account for 1 in 3 deaths in the US. My preliminary findings show that the A-Kinase Anchoring protein AKAP18 sequesters the cAMP-dependent protein kinase (PKA) and the ubiquitin-specific proteinase USP4 with SERCA2. Pilot studies in transfected cells suggest that anchored PKA phosphorylates USP4 to stimulate its deubiquitinase activity. This led to a working hypothesis that both enzymes, when associated with AKAP18, act cooperatively to protect against ubiquitin- mediated removal of SERCA2 from the sarcoplasmic reticulum. This exciting new cardioprotective paradigm will be tested by an experimental approach of two aims. Aim 1 will use state of the art biochemical and biophysical approaches to investigate the molecular organization of the USP4-AKAP18-PKA complex. Mechanistic studies in aim 2 will combine advanced imaging and in vivo genetic strategies to understand how these anchored enzymes coordinately control vital aspects of cardiac physiology, such as calcium handling. My research will be conducted at the University of Washington in the Department of Pharmacology. This environment provides excellent training for academic-track postdoctoral researchers. Training benefits include strong collaboration within and among departments, approachable principal investigators performing innovative work, and frequent seminars from multiple departments featuring world experts in their respective fields. Moreover, the training potential of these studies is high. I will learn cutting edge expansion microscopy imaging techniques and will be introduced to measurement of calcium transients in live cardiomyocytes.