Site-directed mutagenesis of the active site of diacylglycerol kinase α: Calcium and phosphatidylserine stimulate enzyme activity via distinct mechanisms

Takahiro Abe, Xiaolan Lu, Ying Jiang, Clark E. Boccone, Shaomin Qian, Krishna M. Vattem, Ronald C. Wek, James P. Walsh

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

Diacylglycerol kinases (DAGKs) catalyse ATP-dependent phosphorylation of sn-1,2-diacylglycerol that arises during stimulated phosphatidylinositol turnover. DAGKα is activated in vitro by Ca2+ and by acidic phospholipids. The regulatory region of DAGKα includes an N-terminal RVH motif and EF hands that mediate Ca2+-dependent activation. DAGKα also contains tandem C1 protein kinase C homology domains. We utilized yeast, Sacchammyces cerevisiae, which lacks an endogenous DAGK, to express DAGKα and to determine the enzymic activities of different mutant forms of pig DAGKα 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 (Asp 434) or D650 abolished all DAGKα activity, whereas substitution of one among D465, D497, D529 and D697 decreased the activity to 6% or less of that for wild-type DAGKα. 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 DAGKα 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 DAGKα via distinct mechanisms.

Original languageEnglish (US)
Pages (from-to)673-680
Number of pages8
JournalBiochemical Journal
Volume375
Issue number3
DOIs
StatePublished - Nov 1 2003

Keywords

  • Calcium
  • Diacylglycerol
  • Diacylglycerol kinase
  • Phosphatidylserine
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biochemistry

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