Phospholamban, the cardiac sarcoplasmic reticulum proteolipid, is phosphorylated 
by cAMP-dependent protein kinase, by Ca2+/phospholipid-dependent protein kinase, 
and by an endogenous Ca2+/calmodulin-dependent protein kinase, the identity of 
which remains to be defined. The aim of this study was therefore to characterize 
the latter kinase, called phospholamban kinase. Phospholamban kinase was 
purified approximately 42-fold with a yield of 11%. The purified fraction 
exhibits a specific activity of 6.5 nmol of phosphate incorporated into 
exogenous phospholamban per minute per milligram of protein. Phospholamban 
kinase appears to be a high molecular weight enzyme and presents a broad 
substrate specificity, synapsin-1, glycogen synthase, and smooth muscle myosin 
regulatory light chain being the best substrates. Phospholamban kinase 
phosphorylates synapsin-1 on a Mr 30 000 peptide. The enzyme exhibits an optimum 
pH of 8.6, a Km for ATP of 9 microM, and a requirement for Mg2+ ions. These data 
suggest that phospholamban kinase might be an isoenzyme of the multifunctional 
Ca2+/calmodulin-dependent protein kinase. Consequently we have searched for Mr 
50 000-60 000 phosphorylatable subunits among cardiac sarcoplasmic reticulum 
proteins. A Mr 56 000 protein was found to be phosphorylated in the presence of 
Ca2+/calmodulin. Such phosphorylation alters the electrophoretic migration 
velocity of the protein. In addition, this protein that binds calmodulin was 
always found to be present in fractions containing phospholamban kinase 
activity. This Mr 56 000 protein is therefore a good candidate for being a 
subunit of phospholamban kinase. However, the Mr 56 000 calmodulin-binding 
protein and the Mr 53 000 intrinsic glycoprotein which binds ATP are two 
distinct entities.