title: Site-directed mutagenesis of the active site of diacylglycerol kinase alpha: calcium and phosphatidylserine stimulate enzyme activity via distinct mechanisms.
abstract: Diacylglycerol kinases (DAGKs) catalyse ATP-dependent phosphorylation of sn-1,2-diacylglycerol that arises during stimulated phosphatidylinositol turnover. DAGKa is activated in vitro by Ca2+ and by acidic phospholipids. The regulatory region of DAGKa includes an N-terminal RVH motif and EF hands that mediate Ca2+-dependent activation. DAGKa also contains tandem C1 protein kinase C homology domains. We utilized yeast, Saccharomyces cerevisiae, which lacks an endogenous DAGK, to express DAGKa and to determine the enzymic activities of different mutant forms of pig DAGKa in vitro. Six aspartate residues conserved in all DAGKs were individually examined by site-directed mutagenesis. Five of these aspartate residues reside in conserved blocks that correspond to sequences in the catalytic site of phosphofructokinases. Mutation of D434 (Asp434) or D650 abolished all DAGKa activity, whereas substitution of one among D465, D497, D529 and D697 decreased the activity to 6% or less of that for wild-type DAGKa. Roles of homologous residues in phosphofructokinases suggested that the N-terminal half of the DAGK catalytic domain binds Mg-ATP and the C-terminal half binds diacylglycerol. A DAGKa mutant with its entire regulatory region deleted showed a much decreased activity that was not activated by Ca2+, but still exhibited PS (phosphatidylserine)-dependent activation. Moreover, mutations of aspartate residues at the catalytic domain had differential effects on activation by Ca2+ and PS. These results indicate that Ca2+ and PS stimulate DAGKa via distinct mechanisms.
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Drive, MS 4053, Indianapolis, IN 46202, USA.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Abe
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Lu
authors.forename: Ying
authors.initials: Y
authors.lastname: Jiang
authors.forename: Clark E
authors.initials: CE
authors.lastname: Boccone
authors.forename: Shaomin
authors.initials: S
authors.lastname: Qian
authors.forename: Krishna M
authors.initials: KM
authors.lastname: Vattem
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Wek
authors.forename: James P
authors.initials: JP
authors.lastname: Walsh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002118:Calcium / Q000494:pharmacology; D020134:Catalytic Domain / Q000235:genetics; D002522:Chlorocebus aethiops; D019852:Diacylglycerol Kinase / Q000235:genetics* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010718:Phosphatidylserines / Q000494:pharmacology; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013552:Swine
chemical_list: D010718:Phosphatidylserines; D001224:Aspartic Acid; D019852:Diacylglycerol Kinase; D002118:Calcium