Ser-2030, but not Ser-2808, is the major phosphorylation site in cardiac ryanodine receptors responding to protein kinase A activation upon beta-adrenergic stimulation in normal and failing hearts.
We have recently shown that RyR2 (cardiac ryanodine receptor) is phosphorylated by PKA (protein kinase A/cAMP-dependent protein kinase) at two major sites, Ser-2030 and Ser-2808. In the present study, we examined the properties and physiological relevance of phosphorylation of these two sites. Using site- and phospho-specific antibodies, we demonstrated that Ser-2030 of both recombinant and native RyR2 from a number of species was phosphorylated by PKA, indicating that Ser-2030 is a highly conserved PKA site. Furthermore, we found that the phosphorylation of Ser-2030 responded to isoproterenol (isoprenaline) stimulation in rat cardiac myocytes in a concentration- and time-dependent manner, whereas Ser-2808 was already substantially phosphorylated before beta-adrenergic stimulation, and the extent of the increase in Ser-2808 phosphorylation after beta-adrenergic stimulation was much less than that for Ser-2030. Interestingly, the isoproterenol-induced phosphorylation of Ser-2030, but not of Ser-2808, was markedly inhibited by PKI, a specific inhibitor of PKA. The basal phosphorylation of Ser-2808 was also insensitive to PKA inhibition. Moreover, Ser-2808, but not Ser-2030, was stoichiometrically phosphorylated by PKG (protein kinase G). In addition, we found no significant phosphorylation of RyR2 at the Ser-2030 PKA site in failing rat hearts. Importantly, isoproterenol stimulation markedly increased the phosphorylation of Ser-2030, but not of Ser-2808, in failing rat hearts. Taken together, these observations indicate that Ser-2030, but not Ser-2808, is the major PKA phosphorylation site in RyR2 responding to PKA activation upon beta-adrenergic stimulation in both normal and failing hearts, and that RyR2 is not hyperphosphorylated by PKA in heart failure. Our results also suggest that phosphorylation of RyR2 at Ser-2030 may be an important event associated with altered Ca2+ handling and cardiac arrhythmia that is commonly observed in heart failure upon beta-adrenergic stimulation.
Cardiovascular Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta, Canada.
Bailong
B
Xiao
Guofeng
G
Zhong
Masakazu
M
Obayashi
Dongmei
D
Yang
Keyun
K
Chen
Michael P
MP
Walsh
Yakhin
Y
Shimoni
Heping
H
Cheng
Henk
H
Ter Keurs
S R Wayne
SR
Chen
D000318:Adrenergic beta-Agonists / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D001145:Arrhythmias, Cardiac / Q000503:physiopathology; D001596:Benzylamines / Q000494:pharmacology; D015153:Blotting, Western; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000494:pharmacology; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D006333:Heart Failure / Q000188:drug therapy / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D017136:Ion Transport / Q000187:drug effects; D007545:Isoproterenol / Q000494:pharmacology* / Q000627:therapeutic use; D007668:Kidney / Q000166:cytology; D008387:Marine Toxins; D051379:Mice; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism; D010080:Oxazoles / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000502:physiology; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013329:Structure-Activity Relationship; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection; D000096926:Benzenesulfonamides
D000318:Adrenergic beta-Agonists; D001596:Benzylamines; D002352:Carrier Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol; D008387:Marine Toxins; D010080:Oxazoles; D010446:Peptide Fragments; D010749:Phosphoprotein Phosphatases; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D019831:Sodium-Calcium Exchanger; D019311:Staurosporine; D013449:Sulfonamides; C477818:myristoylated protein kinase A inhibitor amide 14-22; C072105:KN 93; D000096926:Benzenesulfonamides; C059041:calyculin A