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 Ca2+-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 Ca2+-free condition, the EF-hand masks the PS-binding site of the DG kinase, and that the Ca2+-binding results in the exposure of the PS-binding site through the conformational change of the EF-hand region. © 1991.