Concerted phosphorylation of the 26-kilodalton phospholamban oligomer and of the low molecular weight phospholamban subunits.
Phospholamban (PLB) from cardiac sarcoplasmic reticulum (SR) was phosphorylated under various conditions by the adenosine cyclic 3',5'-phosphate (cAMP)-dependent and/or the calmodulin-dependent protein kinase. The small shifts in apparent molecular weight resulting from the incorporation of Pi groups in the PLB complexes were analyzed by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In parallel experiments, PLB was dissociated into its subunits and analyzed by using a newly developed isoelectric focusing system. The pI values of the PLB subunits phosphorylated by the cAMP- or calmodulin-dependent kinase were 6.2 and 6.4, respectively. Double phosphorylation of the same subunit resulted in an acidic shift of the pI to 5.2. The combined analysis of the behavior of the PLB complex and of its subunits has greatly simplified the interpretation of the complex phosphorylation pattern and has led to the following conclusions: The PLB complex is composed of five probably identical subunits, each of them containing a distinct phosphorylation site for the calmodulin- and the cAMP-dependent kinase. The population of PLB interacting with the endogenous calmodulin-dependent kinase cannot be phosphorylated by the cAMP-dependent kinase unless previously phosphorylated in the presence of calmodulin. It was also observed that after maximal phosphorylation of PLB in the presence of very large amounts of the cAMP-dependent protein kinase, the Ca2+ pumping rate of the cardiac SR ATPase is stimulated up to 5-fold, i.e., a level of a stimulation which exceeds considerably the values so far reported in the literature.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004285:Dogs; D006352:Heart Ventricles / Q000201:enzymology; D046911:Macromolecular Substances; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D002135:Calcium-Binding Proteins; D046911:Macromolecular Substances; D000099211:Phospholamban