Expression and site-specific mutagenesis of phospholamban. Studies of residues involved in phosphorylation and pentamer formation.
Full-length cDNAs encoding either dog cardiac or rabbit skeletal muscle phospholamban were expressed transiently in COS-1 cells. The expressed protein displayed the mobility of a pentamer when dissolved in sodium dodecyl sulfate and separated in polyacrylamide gels, and of a monomer when boiled prior to polyacrylamide gel separation. Site-specific mutagenesis was used to analyze the roles of several amino acids in the structure and function of the protein. Ser16 and Thr17 were shown to be phosphorylated uniquely by cAMP- and calmodulin-dependent protein kinases, respectively, confirming earlier observations on the native protein (Simmerman, H. K. B., Collins, J. H., Theibert, J.L., Wegener, A.D., and Jones, L.R. (1986) J. Biol. Chem. 261, 13333-13341). Arg13 and Arg14 were shown to be essential for both types of phosphorylation, and Arg9 was shown to be essential for calmodulin-dependent phosphorylation. In studies of pentamer stability, mutation of Gln22-Gln23 to Ala-Ala or Glu-Glu, of Gln26-Asn27 to Glu-Asp, or of Gln29-Asn30 to Glu-Asp had no effect on thermal stability of the pentamer, suggesting that hydrogen bonding involving these residues in domain IB is not important for pentamer stability. By contrast, mutation of Cys36, Cys41, and Cys46 in transmembrane domain II to Ser, Ala, or Phe diminished the stability of the pentamer when microsomal proteins were dissociated in sodium dodecyl sulfate and separated by polyacrylamide gel electrophoresis. In particular, the Cys41 to Phe mutant existed as a monomer at ambient temperature. These results suggest that the intramembranous cysteine residues are important for pentamer formation even though they are not disulfide-bonded.
Fujii
J
J
Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Maruyama
K
K
Tada
M
M
MacLennan
D H
DH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002147:Calmodulin / Q000502:physiology; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D003545:Cysteine / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D004247:DNA / Q000302:isolation & purification; D004285:Dogs; D007668:Kidney; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000502:physiology; D012694:Serine / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D013912:Threonine / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D000099211:Phospholamban
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D003545:Cysteine; D004247:DNA; D011494:Protein Kinases; D012694:Serine; D013912:Threonine; D000099211:Phospholamban