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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
5F31GM087079-03
Application #
8010190
Study Section
Special Emphasis Panel (ZRG1-GGG-F (29))
Program Officer
Gaillard, Shawn R
Project Start
2009-01-16
Project End
2012-01-15
Budget Start
2011-01-16
Budget End
2012-01-15
Support Year
3
Fiscal Year
2011
Total Cost
$41,800
Indirect Cost
Name
Johns Hopkins University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Depry, Charlene; Mehta, Sohum; Li, Ruojing et al. (2015) Visualization of Compartmentalized Kinase Activity Dynamics Using Adaptable BimKARs. Chem Biol 22:1470-1479
Chakir, Khalid; Depry, Charlene; Dimaano, Veronica L et al. (2011) Galphas-biased beta2-adrenergic receptor signaling from restoring synchronous contraction in the failing heart. Sci Transl Med 3:100ra88
Gao, Xinxin; Lowry, Pamela R; Zhou, Xin et al. (2011) PI3K/Akt signaling requires spatial compartmentalization in plasma membrane microdomains. Proc Natl Acad Sci U S A 108:14509-14
Depry, Charlene; Allen, Michael D; Zhang, Jin (2011) Visualization of PKA activity in plasma membrane microdomains. Mol Biosyst 7:52-8