title: Systems analysis of PKA-mediated phosphorylation gradients in live cardiac myocytes.
abstract: Compartmentation and dynamics of cAMP and PKA signaling are important determinants of specificity among cAMP's myriad cellular roles. Both cardiac inotropy and the progression of heart disease are affected by spatiotemporal variations in cAMP/PKA signaling, yet the dynamic patterns of PKA-mediated phosphorylation that influence differential responses to agonists have not been characterized. We performed live-cell imaging and systems modeling of PKA-mediated phosphorylation in neonatal cardiac myocytes in response to G-protein coupled receptor stimuli and UV photolysis of "caged" cAMP. cAMP accumulation was rate-limiting in PKA-mediated phosphorylation downstream of the beta-adrenergic receptor. Prostaglandin E1 stimulated higher PKA activity in the cytosol than at the sarcolemma, whereas isoproterenol triggered faster sarcolemmal responses than cytosolic, likely due to restricted cAMP diffusion from submembrane compartments. Localized UV photolysis of caged cAMP triggered gradients of PKA-mediated phosphorylation, enhanced by phosphodiesterase activity and PKA-mediated buffering of cAMP. These findings indicate that combining live-cell FRET imaging and mechanistic computational models can provide quantitative understanding of spatiotemporal signaling.
authors.affiliation: Department of Bioengineering, Whitaker Institute of Biomedical Engineering, University of California at San Diego, La Jolla, CA 92093, USA.
authors.forename: Jeffrey J
authors.initials: JJ
authors.lastname: Saucerman
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.forename: Jody C
authors.initials: JC
authors.lastname: Martin
authors.forename: Lili X
authors.initials: LX
authors.lastname: Peng
authors.forename: Antine E
authors.initials: AE
authors.lastname: Stenbit
authors.forename: Roger Y
authors.initials: RY
authors.lastname: Tsien
authors.forename: Andrew D
authors.initials: AD
authors.lastname: McCulloch
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002462:Cell Membrane / Q000201:enzymology; D002470:Cell Survival; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D007700:Kinetics; D008954:Models, Biological; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases