Subunit structure and multiple phosphorylation sites of phospholamban.
The phosphorylation-induced mobility shift of the high molecular weight form of phospholamban (24,500 daltons) in the cardiac sarcoplasmic reticulum produced on 3',5'-cyclic AMP (cAMP)-dependent phosphorylation with 5 mM ATP was resolved into five clear steps on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and on Ca2+-calmodulin-dependent phosphorylation into ten steps. The mobility shift of the low molecular weight form of phospholamban (less than 14,400 daltons) in these reactions occurred in one step and two steps, respectively. With the two protein kinase activities, the electrophoretic pattern of the mobility shifts of the high and low molecular weight forms of phospholamban was similar to that obtained with Ca2+-calmodulin-dependent protein kinase alone. The results of pulse-chase experiments involving the centrifuge column method suggested that the site(s) of phosphorylation by cAMP- and Ca2+-calmodulin-dependent protein kinase activities are on the same phospholamban molecule. Two-dimensional tryptic peptide maps of phosphorylated phospholamban indicated that cAMP-dependent protein kinase phosphorylates at a single site, A, and Ca2+-calmodulin-dependent protein kinase phosphorylates at sites C1 and C2 in the low molecular weight form, where A is different from C1 but may be the same as C2. The high molecular weight form of phospholamban is suggested to be a pentamer of identical monomers (low molecular weight form) having one phosphorylation site for cAMP-dependent protein kinase and two for Ca2+-calmodulin-dependent protein kinase.
D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008970:Molecular Weight; D009206:Myocardium / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D014357:Trypsin; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D010446:Peptide Fragments; D011494:Protein Kinases; D014357:Trypsin; D000099211:Phospholamban