Phosphorylation of the purified cardiac ryanodine receptor by exogenous and endogenous protein kinases.
The ryanodine receptor is the main Ca(2+)-release structure in skeletal and cardiac sarcoplasmic reticulum. In both tissues, phosphorylation of the ryanodine receptor has been proposed to be involved in the regulation of Ca2+ release. In the present study, we have examined the ability of the purified cardiac ryanodine receptor to serve as a substrate for phosphorylation by exogenously added catalytic subunit of the cyclic AMP (cAMP)-dependent protein kinase (PK-A), cyclic GMP (cGMP)-dependent protein kinase (PK-G), or calmodulin-dependent protein kinase (PK-CaM). A large amount of phosphate incorporation was observed for PK-CaM (938 +/- 48 pmol of Pi/mg of purified channel protein), whereas the level of phosphorylation was considerably lower with PK-A or PK-G (345 +/- 139 and 96 +/- 6 pmol/mg respectively). In addition, endogenous PK-CaM activity co-migrates with the ryanodine receptor through several steps of purification, suggesting a strong association of the two proteins. This endogenous PK-CaM activity is abolished by a PK-CaM-specific synthetic peptide inhibitor. Endogenous cAMP- and cGMP-dependent phosphorylation was not observed in the purified ryanodine-receptor preparation. Taken together, these observations imply that PK-CaM is the physiologically relevant protein kinase, capable of phosphorylating the channel protein to a minimum stoichiometry of 2 mol of Pi per mol of tetramer.
Hohenegger
M
M
Institute of Pharmacology, University of Vienna, Austria.
Suko
J
J
D000818:Animals; D015220:Calcium Channels / Q000302:isolation & purification / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods; D002846:Chromatography, Affinity; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D009124:Muscle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D012433:Ryanodine / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D015220:Calcium Channels; D002147:Calmodulin; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D007371:Interferon-gamma; D011494:Protein Kinases; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases