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.