Dependence of cardiac sarcoplasmic reticulum calcium pump activity on the phosphorylation status of phospholamban.
The application of electrophoretic resolution of the different phosphorylation species of pentameric phospholamban as a measure of phosphorylation stoichiometry was examined and verified. This enabled a critical evaluation of a number of issues central to current models of calcium pump regulation in cardiac sarcoplasmic reticulum. The phospholamban content of numerous preparations was calculated from 32P incorporation at a given stoichiometry, and compared with the respective calcium pump concentration (derived by comparison with a Coomassie-stained calibration curve of the fast-twitch skeletal muscle isozyme). A relationship of 2 mol of phospholamban:1 mol of ATPase resulted (phospholamban monomer:ATPase monomer), which was maintained throughout all vesicle subpopulations. The precise mechanism of coupling of phospholamban phosphorylation to calcium pump stimulation was probed, with particular emphasis on the individual contributions of each phosphorylated species (P1 to P5). This relationship could be adequately explained in three ways: (i) each phosphorylation event contributed equally to calcium pump stimulation; (ii) P1 and P2 were incapable of stimulating calcium pump activity, but full stimulation occurred upon generation of species P3; or (iii) the phosphospecies P1 was without effect on basal calcium pump activity, but successive phosphorylations contributed equally to stimulation. Finally, the functional implication of dual site phosphorylation of phospholamban (cAMP- and the endogenous calmodulin-dependent kinases) was examined. No change in calcium pump activity accompanied the second tier of phosphorylation over that achieved by the first.
Colyer
J
J
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Wang
J H
JH
D001693:Biological Transport, Active; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D000099211:Phospholamban
D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D000099211:Phospholamban