Characterization of a novel PKA phosphorylation site, serine-2030, reveals no PKA hyperphosphorylation of the cardiac ryanodine receptor in canine heart failure.
Hyperphosphorylation of the cardiac Ca2+ release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) at serine-2808 has been proposed to be a key mechanism responsible for cardiac dysfunction in heart failure (HF). However, the sites of PKA phosphorylation in RyR2 and their phosphorylation status in HF are not well defined. Here we used various approaches to investigate the phosphorylation of RyR2 by PKA. Mutating serine-2808, which was thought to be the only PKA phosphorylation site in RyR2, did not abolish the phosphorylation of RyR2 by PKA. Two-dimensional phosphopeptide mapping revealed two major PKA phosphopeptides, one of which corresponded to the known serine-2808 site. Another, novel, PKA phosphorylation site, serine 2030, was identified by Edman sequencing. Using phospho-specific antibodies, we showed that the novel serine-2030 site was phosphorylated in rat cardiac myocytes stimulated with isoproterenol, but not in unstimulated cells, whereas serine-2808 was considerably phosphorylated before and after isoproterenol treatment. We further showed that serine-2030 was stoichiometrically phosphorylated by PKA, but not by CaMKII, and that mutations of serine-2030 altered neither the FKBP12.6-RyR2 interaction nor the Ca2+ dependence of [3H]ryanodine binding. Moreover, the levels of phosphorylation of RyR2 at serine-2030 and serine-2808 in both failing and non-failing canine hearts were similar. Together, our data indicate that serine-2030 is a major PKA phosphorylation site in RyR2 responding to acute beta-adrenergic stimulation, and that RyR2 is not hyperphosphorylated by PKA in canine HF.
Xiao
Bailong
B
Cardiovascular Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, AB, Canada.
Jiang
Ming Tao
MT
Zhao
Mingcai
M
Yang
Dongmei
D
Sutherland
Cindy
C
Lai
F Anthony
FA
Walsh
Michael P
MP
Warltier
David C
DC
Cheng
Heping
H
Chen
S R Wayne
SR
D000818:Animals; D002118:Calcium / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004285:Dogs; D006333:Heart Failure / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D032383:Myocytes, Cardiac / Q000378:metabolism; D010766:Phosphorylation; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012694:Serine / Q000378:metabolism
D019837:Ryanodine Receptor Calcium Release Channel; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D007545:Isoproterenol; D002118:Calcium