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 chara...

Full description

Saved in:
Bibliographic Details
Published in:The Journal of biological chemistry 2004-02, Vol.279 (9), p.8230-8241
Main Authors: Gómez-Merino, Fernando C., Brearley, Charles A., Ornatowska, Magdalena, Abdel-Haliem, Mahmoud E.F., Zanor, María-Inés, Mueller-Roeber, Bernd
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Arabidopsis - enzymology
Arabidopsis - genetics
Arabidopsis thaliana
Cold Temperature
diacylglycerol
Diacylglycerol Kinase - chemistry
Diacylglycerol Kinase - genetics
Diacylglycerol Kinase - metabolism
Diglycerides - metabolism
DNA, Complementary - isolation & purification
DNA, Plant - isolation & purification
Flowers - enzymology
Gene Expression
Hydrogen-Ion Concentration
Isoenzymes - chemistry
Kinetics
Magnesium - pharmacology
Molecular Sequence Data
phosphatidic acid
Phosphorylation
Phylogeny
Plant Leaves - enzymology
Plant Roots - embryology
Signal Transduction
Substrate Specificity
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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 ∼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 Km and Vmax values were 125 μm for DAG and 0.25 pmol of PA min-1 μg-1, respectively. The enzyme was maximally active at pH 7.2. Its activity was Mg2+-dependent and affected by the presence of detergents, salts, and the DGK inhibitor R59022, but not by Ca2+. 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 °C), pointing to a role in cold signal transduction.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M312187200