Localization and functional role of the calmodulin-binding domain of phospholamban in cardiac sarcoplasmic reticulum vesicles.

Limited proteolysis and affinity-labeling techniques have been used to localize the calmodulin-binding domain of phospholamban, the major substrate for both cAMP- and calmodulin-dependent protein kinases in cardiac sarcoplasmic reticulum (SR). SR vesicles, treated with increasing concentrations of trypsin (likely hydrolyzing at Arg-25 in the cytoplasmic region of phospholamban), exhibited a subsequent loss of both cAMP- and calmodulin-dependent phosphorylation, as well as calmodulin affinity-labeling of phospholamban. When SR vesicles were treated with increasing concentrations of chymotrypsin (which likely cleaves at Tyr-6 of phospholamban) there was no effect on the cAMP-dependent phosphorylation of phospholamban. However, similar concentrations of chymotrypsin resulted in a loss of both calmodulin affinity-labeling and calmodulin-dependent phosphorylation of phospholamban (at Thr-17). When SR vesicles were treated with increasing concentrations of Endoproteinase Lys-C (which hydrolyzes phospholamban at Lys-3) both the calmodulin affinity-labeling and the calmodulin-dependent, but not the cAMP-dependent, phosphorylation of phospholamban were inhibited. These data were complemented by 1H-NMR studies on the complex formed by calmodulin and a phospholamban peptide. These data suggest that binding of calmodulin to phospholamban may be an essential intermediate step in the calmodulin-dependent phosphorylation of phospholamban.