Nature and site of phospholamban regulation of the Ca2+ pump of sarcoplasmic reticulum.
The rapid removal of Ca2+ ions from the cytosol, necessary for the efficient relaxation of cardiac muscle cells, is performed by the Ca2+-pumping ATPase of the sarcoplasmic reticulum. The calcium pump is activated by cyclic AMP- and calmodulin-dependent phosphorylation of phospholamban, an integral membrane protein of the sarcoplasmic reticulum. Using a heterobifunctional crosslinking agent which can be cleaved and photoactivated, we provide evidence for a direct interaction between the two proteins. Only the non-phosphorylated form of phospholamban interacts with the ATPase, demonstrating that phospholamban is an endogenous inhibitor that is removed from the ATPase by phosphorylation. Non-phosphorylated phospholamban interacts only with the calcium-free conformation of the ATPase and is released when it is converted to the calcium-bound state. We localized the site of interaction to a single peptide isolated after cyanogen bromide cleavage of the ATPase. The peptide derives from a domain just C-terminal to the aspartyl phosphate of the active site. This domain is unique to ATPases of the sarcoplasmic reticulum in that it has no homology with any other phosphorylation-type ion pump. The domain occurs in both slow- and fast-twitch isoforms of the ATPase, even though phospholamban is not expressed in fast-twitch muscles.
James
P
P
Laboratory of Biochemistry, Swiss Federal Institute of Technology, Zürich.
Inui
M
M
Tada
M
M
Chiesi
M
M
Carafoli
E
E
D000251:Adenosine Triphosphatases / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004285:Dogs; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012689:Sequence Homology, Nucleic Acid; D012756:Sheep; D000099211:Phospholamban
D000251:Adenosine Triphosphatases; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D010446:Peptide Fragments; D000099211:Phospholamban