Regulation of the cardiac ryanodine receptor by protein kinase-dependent phosphorylation.
The exogenous addition of the catalytic subunit of cAMP-dependent protein kinase (PKA), cGMP-dependent protein kinase (PKG), or calmodulin (CaM) induced rapid phosphorylation of the ryanodine receptor (Ca2+ release channel) in canine cardiac microsomes treated with 1 mM [gamma-32P]ATP. Added protein kinase C (PKC) also phosphorylated the cardiac ryanodine receptor but at a relatively slow rate. The observed level of PKA-, PKG-, or PKC-dependent phosphorylation of the ryanodine receptor was comparable to the maximum level of [3H]ryanodine binding in cardiac microsomes, whereas the level of CaM-dependent phosphorylation was about 4 times greater. Phosphorylation by PKA, PKG, and PKC increased [3H]ryanodine binding in cardiac microsomes by 22 +/- 5, 17 +/- 4, and 15 +/- 9% (average +/- SD, n = 4-5), respectively. In contrast, incubation of microsomes with 5 microM CaM alone and 5 microM CaM plus 1 mM ATP decreased [3H]ryanodine binding by 38 +/- 14 and 53 +/- 15% (average +/- SD, n = 6), respectively. Phosphopeptide mapping and phosphoamino acid analysis provided evidence suggesting that PKA, PKG, and PKC predominantly phosphorylate serine residue(s) in the same phosphopeptide (peptide 1), whereas the endogenous CaM-kinase phosphorylates serine residue(s) in a different phosphopeptide (peptide 4). Photoaffinity labeling of microsomes with photoreactive 125I-labeled CaM revealed that CaM bound to a high molecular weight protein, which was immunoprecipitated by a monoclonal antibody against the cardiac ryanodine receptor. These results suggest that protein kinase-dependent phosphorylation and CaM play important regulatory roles in the function of the cardiac sarcoplasmic reticulum Ca2+ release channel.
Takasago
T
T
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka.
Imagawa
T
T
Furukawa
K
K
Ogurusu
T
T
Shigekawa
M
M
D000345:Affinity Labels; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D004285:Dogs; D066298:In Vitro Techniques; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011950:Receptors, Cholinergic / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel
D000345:Affinity Labels; D015220:Calcium Channels; D002147:Calmodulin; D011950:Receptors, Cholinergic; D019837:Ryanodine Receptor Calcium Release Channel; D011494:Protein Kinases