Phosphatases, local structures and signaling in heart

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DESCRIPTION (provided by applicant): Local signaling in heart includes a kinase-phosphatase balance that controls the phosphorylation state of functional proteins. As studies on kinases can only reveal part of the signaling cascade, the importance of phosphatases is underappreciated. New results by the Project Leader show that inhibition of serine/threonine phosphatases, PP1 and PP2A, lead to increases in L-type lCa and mislocalization of a t-tubule cytoskeletal protein. Also, the PI has found that B-targeting subunits of PP2A are segregated to strategic sites within ventricular myocytes, including striated structures and focal regions of the plasma membrane. Thus, this project will define the links between phosphatase subcellular targeting, cytoskeletal architecture, and Ca2+ signaling in cardiac myocytes. The aims are a series of interrelated experiments that are co-mingled with the efforts of colleagues on this PPG. Aim 1 will define the functional sites to which the phosphatase PP2A is targeted in cardiac myocytes. This series will use a combination of intact cell studies combined with molecular methods. Aim 2 will build on the results of (1) and will identify the specific amino acid sequence motifs in PP2A B-targeting subunits that determine its segregation to discrete cardiac cell structures. Virus directed gene transfer of GFP fusion proteins will be used in the analysis. Aim 3 will define the role of phosphatases and t-tubule cytoskeleton proteins in the regulation of L-type Ca2+ channel activity. This series will examine the local kinase/ phosphatase balance in normal and heart failure animal models developed in Projects 2 and 3. The role of t-tubule cytoskeletal structure in control of ICa will be examined with novel dominant negative probes developed in Project 2. Aim 4 will provide a functional link to Aims (1) and (2) above, and will examine how alterations in PP2A targeting change signal transduction cascades. This series will capitalize on intriguing new results that PP2A B subunit overexpression leads to a blunted beta-adrenergic response in cultured cardiac myocytes. The precise molecular analysis will be complemented by studies in cells from stress-activated models of heart failure developed in Project 3, where dramatic increases in these B subunits are also seen. This project will use a series of complementary approaches including voltage clamp, high resolution confocal imaging, adenoviral gene transfer, and biochemical analyses to critically examine the links between local phosphorylation, cytoskeletal structures, and Ca2+ signaling in heart. Another strength of this project is that the flexible plans will capitalize on the results from all of the Project Leaders in the PPG.