Identification and gene-targeting of a novel diacylglycerol kinase highly selective for arachidonate-containing substrates
Creators: name: Tang, Wen
Publisher: name: University of Utah
Year: 2012

description: Diacylglycerol (DAG) is an important second messenger and occupies a central position in the biosynthesis of phospholipids and tnacylglycerols. Conversion of DAG to phosphatidic acid by diacylglycerol kinase (DGK) regulates the intracellular concentration of DAG and the route it takes.  A novel human DGK was cloned by polymerase chain reaction (PCR) using degenerate primers. This enzyme, designated hDGKe, contains 566 residues with a predicted molecular weight of 64 kDa. hDGKe has the simplest structure among the known DGKs: two distinctive cysteine-rich domains (CRDs) in its N-terminal region and a catalytic domain in its C-terminal region. Northern blotting demonstrated that hDGKe is expressed predominantly in testis. COS-7 cells transfected with the hDGKe expressed a 58 kDa protein and had a marked increase in DGK activity. A unique feature of hDGKe is that it demonstrated clear selectivity for arachidonoyl-containing species of diacylglycerol.  PCR based site-directed mutagenesis was used to map the regions in hDGKe that are responsible for substrate selectivity. Chimeric molecules were constructed from hDGKe and hDGK£, a DGK without substrate selectivity, by domain-exchange. Analysis of the enzyme activity and substrate selectivity of these chimeric molecules and a truncated hDGKe that lacked CRDs showed that the CRDs of hDGKe are not sufficient to confer substrate selectivity to DGKe although they are necessary for maintaining hDGKe enzyme activity; the two subcatalytic domains in hDGKe and hDGK£ appear to interact with each other to exert their catalytic function. Mutagenesis studies of the first CRD in hDGKe revealed that the position of its last cysteine was crucial in maintaining the DGK activity of hDGKe although it was unlikely the DAG binding site. Mutations of some unique residues in the catalytic domain of hDGKe, however, did not affect substrate selectivity. A glycine-rich motif in hDGKe was also identified as the potential ATP binding site by site-directed mutagenesis.  Analysis of the organization of the hDGKe gene revealed that it contains at least 12 exons spanning approximately 30 kb of genomic sequence. The hDGKe gene was mapped to chromosome 17q22, the same region where the eighth locus of autosomal dominant retinitis pigmentosa (adRP) resides. Analysis by Northern blotting showed that hDGKe was expressed in human retina which suggested it as a candidate for adRP. The hDGKe gene was localized to a YAC clones that also contained an STS marker in the eighth adRP locus. Direct sequencing following PCR amplification of DNA samples from two type 8 adRP patients, however, did not reveal any mutation in hDGKe exons. The hDGKe gene is thus unlikely to be an adRP gene.  Cloned murine DGKe displayed similar selectivity for arachidonate-containing substrates as hDGKe. Northern blotting demonstrated that murine DGKe mRNA had highest expression level in brain. The mRNA of murine DGKe was detected by in situ hybridization in the Purkinje cells of cerebellum, the pyramidal cells in the CA3 region of hippocampus, and many other neurons in brain involved in motor and sensory functions. Mice with disrupted DGKe alleles were generated to study the functions of DGKe in vivo. DGKe gene knockout mice are viable and display normal feeding, grooming, and mating behavior. Interestingly, male DGKe mutant mice exhibited impaired motor coordination. DGKe therefore appears to play an important role in neurologicalfunction.