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.
Strasburg
G M
GM
Department of Food Science and Human Nutrition, Michigan State University, East Lansing.
Hanson
T P
TP
Ouyang
H X
HX
Louis
C F
CF
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002918:Chymotrypsin; D004285:Dogs; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013552:Swine; D014357:Trypsin; D000099211:Phospholamban
D000345:Affinity Labels; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002918:Chymotrypsin; D008666:Metalloendopeptidases; D014357:Trypsin; D000099211:Phospholamban; C039830:peptidyl-Lys metalloendopeptidase