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 su bstrate for both cAMP- and calmodulin-dependent protein kinases in car diac sarcoplasmic reticulum (SR). SR vesicles, treated with increasing concentrations of trypsin (likely hydrolyzing at Arg-25 in the cytopl asmic region of phospholamban), exhibited a subsequent loss of both cA MP- and calmodulin-dependent phosphorylation, as well as calmodulin af finity-labeling of phospholamban. When SR vesicles were treated with i ncreasing concentrations of chymotrypsin (which likely cleaves at Tyr- 6 of phospholamban) there was no effect on the cAMP-dependent phosphor ylation of phospholamban. However, similar concentrations of chymotryp sin resulted in a loss of both calmodulin affinity-labeling and calmod ulin-dependent phosphorylation of phospholamban (at Thr-17). When SR v esicles were treated with increasing concentrations of Endoproteinase Lys-C (which hydrolyzes phospholamban at Lys-3) both the calmodulin af finity-labeling and the calmodulin-dependent, but not the cAMP-depende nt, phosphorylation of phospholamban were inhibited. These data were c omplemented by H-1-NMR studies on the complex formed by calmodulin and a phospholamban peptide. These data suggest that binding of calmoduli n to phospholamban may be an essential intermediate step in the calmod ulin-dependent phosphorylation of phospholamban.