Phospholamban is the major membrane protein of the heart phosphorylated in 
response to beta-adrenergic stimulation. In cell-free systems, cAMP-dependent 
protein kinase catalyzes exclusive phosphorylation of serine 16 of 
phospholamban, whereas Ca2+/calmodulin-dependent protein kinase gives exclusive 
phosphorylation of threonine 17 (Simmerman, H. K. B., Collins, J. H., Theibert, 
J. L., Wegener, A. D., and Jones, L. R. (1986) J. Biol. Chem. 261, 13333-13341). 
In this work we have localized the sites of phospholamban phosphorylation in 
intact ventricles treated with the beta-adrenergic agonist isoproterenol. 
Isolation of phosphorylated phospholamban from 32P-perfused guinea pig 
ventricles, followed by partial acid hydrolysis and phosphoamino acid analysis, 
revealed phosphorylation of both serine and threonine residues. At steady state 
after isoproterenol exposure, phospholamban contained approximately equimolar 
amounts of these two phosphoamino acids. Two major tryptic phosphopeptides 
containing greater than 90% of the incorporated radioactivity were obtained from 
phospholamban labeled in intact ventricles. The amino acid sequences of these 
two tryptic peptides corresponded exactly to residues 14-25 and 15-25 of canine 
cardiac phospholamban, thus localizing the sites of in situ phosphorylation to 
serine 16 and threonine 17. Phosphorylation of phospholamban at two sites in 
heart perfused with isoproterenol was supported by detection of 11 distinct 
mobility forms of the pentameric protein by use of the Western blotting method, 
consistent with each phospholamban monomer containing two phosphorylation sites, 
and with each pentamer containing from 0 to 10 incorporated phosphates. Our 
results localize the sites of in situ phospholamban phosphorylation to serine 16 
and threonine 17 and, furthermore, are consistent with the phosphorylations of 
these 2 residues being catalyzed by cAMP- and Ca2+/calmodulin-dependent protein 
kinases, respectively.