The regulatory role of EF-hand motifs of pig 80K diacylglycerol kinase as assessed using truncation and deletion mutants.
To elucidate the regulatory function of EF-hand motifs of pig 80K diacylglycerol (DG) kinase, we constructed and expressed several truncation and deletion mutants of the enzyme in E. coli or COS-7 cells. The bacterially expressed EF-hand region could bind Ca2+ and was suggested to undergo conformational change like calmodulin. A mutant enzyme lacking EF-hands lost Ca(2+)-binding activity, but could be fully activated by phosphatidylserine (PS) or deoxycholate in the absence of Ca2+. The full activation of the wild-type enzyme by PS, on the other hand, was totally dependent on Ca2+. Further, the wild-type enzyme expressed in COS-7 cells was exclusively soluble, whereas the EF-hand-deleted mutant was considerably associated with the membranes. The results suggest that under Ca(2+)-free condition, the EF-hand masks the PS-binding site of the DG kinase, and that the Ca(2+)-binding results in the exposure of the PS-binding site through the conformational change of the EF-hand region.
Sakane
F
F
Department of Biochemistry, Sapporo Medical College, Japan.
Imai
S
S
Yamada
K
K
Kanoh
H
H
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D019852:Diacylglycerol Kinase; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010770:Phosphotransferases / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013552:Swine; D014162:Transfection
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D010770:Phosphotransferases; D019852:Diacylglycerol Kinase