Heterogeneous distribution of calmodulin- and cAMP-dependent regulation of Ca2+ uptake in cardiac sarcoplasmic reticulum subfractions.
The activity of the Ca2+-pumping ATPase of cardiac sarcoplasmic reticulum is controlled by the phosphorylation level of the intrinsic membrane protein phospholamban. Phospholamban monomers contain two distinct phosphorylation sites for either the cAMP-dependent or the calmodulin-dependent kinase. The two kinases, however, preferentially phosphorylate different populations of phospholamban molecules and double phosphorylation of the same subunit by their concerted action is a phenomenon that occurs only under particular experimental conditions. This study investigates the phosphorylation pattern of phospholamban in various subfractions derived from dog cardiac sarcoplasmic reticulum. The results show that the endogenous calmodulin-dependent kinase preferentially phosphorylates the phospholamban population found in association with the cisternal compartments of the reticulum. The differential phosphorylation occurs despite the presence of sufficient amounts of the kinase in all sarcoplasmic reticulum subfractions. On the other hand, phospholamban molecules localized on the longitudinal system are preferential substrates for the cAMP-dependent kinase. Possibly, the different lipid and/or protein microenvironment of phospholamban in the various sarcoplasmic reticulum domains is responsible for the apparent heterogeneity of phosphorylation. The present findings are compatible with the concept of additive and independent action of the cAMP-dependent and calmodulin-dependent kinases on cardiac sarcoplasmic reticulum. The imply, however, that different regions of the sarcoplasmic reticulum network are controlled by the two regulatory mechanisms.
Gasser
J
J
Laboratory of Biochemistry, Swiss Federal Institute of Technology, Zürich.
Paganetti
P
P
Carafoli
E
E
Chiesi
M
M
D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D054736:Elongation Factor 2 Kinase; D009206:Myocardium / Q000201:enzymology / Q000378:metabolism; D010710:Phosphates / Q000032:analysis; D011494:Protein Kinases / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000252:Calcium-Transporting ATPases; D002147:Calmodulin; D054736:Elongation Factor 2 Kinase; D010710:Phosphates; D011494:Protein Kinases; D000099211:Phospholamban