AtDGK2, a novel diacylglycerol kinase from Arabidopsis thaliana, phosphorylates 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol and exhibits cold-inducible gene expression.
Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to generate phosphatidic acid (PA). Both DAG and PA are implicated in signal transduction pathways. DGKs have been widely studied in animals, but their analysis in plants is fragmentary. Here, we report the cloning and biochemical characterization of AtDGK2, encoding DGK from Arabidopsis thaliana. AtDGK2 has a predicted molecular mass of 79.4 kDa and, like AtDGK1 previously reported, harbors two copies of a phorbol ester/DAG-binding domain in its N-terminal region. AtDGK2 belongs to a family of seven DGK genes in A. thaliana. AtDGK3 to AtDGK7 encode approximately 55-kDa DGKs that lack a typical phorbol ester/DAG-binding domain. Phylogenetically, plant DGKs fall into three clusters. Members of all three clusters are widely expressed in vascular plants. Recombinant AtDGK2 was expressed in Escherichia coli and biochemically characterized. The enzyme phosphorylated 1,2-dioleoyl-sn-glycerol to yield PA, exhibiting Michaelis-Menten type kinetics. Estimated K(m) and V(max) values were 125 microm for DAG and 0.25 pmol of PA min(-1) microg(-1), respectively. The enzyme was maximally active at pH 7.2. Its activity was Mg(2+)-dependent and affected by the presence of detergents, salts, and the DGK inhibitor R59022, but not by Ca(2+). AtDGK2 exhibited substrate preference for unsaturated DAG analogues (i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol and 1,2-dioleoyl-sn-glycerol). The AtDGK2 gene is expressed in various tissues of the Arabidopsis plant, including leaves, roots, and flowers, as shown by Northern blot analysis and promoter-reporter gene fusions. We found that AtDGK2 is induced by exposure to low temperature (4 degrees C), pointing to a role in cold signal transduction.
Gómez-Merino
Fernando C
FC
University of Potsdam, Institute of Biochemistry and Biology, Karl-Liebknecht-Strasse 24-25, Haus 20, D-14476 Golm/Potsdam, Germany.
Brearley
Charles A
CA
Ornatowska
Magdalena
M
Abdel-Haliem
Mahmoud E F
ME
Zanor
María-Inés
MI
Mueller-Roeber
Bernd
B
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D003080:Cold Temperature; D018076:DNA, Complementary / Q000302:isolation & purification; D018744:DNA, Plant / Q000302:isolation & purification; D019852:Diacylglycerol Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004075:Diglycerides / Q000378:metabolism*; D035264:Flowers / Q000201:enzymology; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010802:Phylogeny; D018515:Plant Leaves / Q000201:enzymology; D018517:Plant Roots / Q000196:embryology; D015398:Signal Transduction; D013379:Substrate Specificity
D018076:DNA, Complementary; D018744:DNA, Plant; D004075:Diglycerides; D007527:Isoenzymes; C075809:1-stearoyl-2-arachidonoylglycerol; D019852:Diacylglycerol Kinase; D008274:Magnesium; C013965:diolein