Molecular Cloning and Characterization of a Novel Human Diacylglycerol Kinase ζ

Abstract

To further explore the role of diacylglycerol metabolism in endothelial responses, we used a degenerate reverse transcription-polymerase chain reaction method to identify diacylglycerol kinase isozymes expressed by human endothelial cells. We report the isolation of a 3.5-kilobase cDNA encoding a novel diacylglycerol kinase (hDGK) with a predicted molecular mass of 103.9 kDa. Human DGK contains two zinc fingers, an ATP binding site, and four ankyrin repeats near the carboxyl terminus. A unique feature, as compared with other diacylglycerol kinases, is the presence of a sequence homologous to the MARCKS phosphorylation site domain. From Northern blot analysis of multiple tissues, we observed that hDGK mRNA is expressed at highest levels in brain. COS-7 cells transfected with the hDGK cDNA express 117-kDa and 114-kDa proteins that react specifically with an antibody to a peptide derived from a unique sequence in hDGK. The transfected cells also express increased diacylglycerol kinase activity, which is not altered in the presence of R59949, an inhibitor of human platelet DGK activity. The hDGK displays stereoselectivity for 1,2-diacylglycerol species in comparison to 1,3-diacylglycerol, but does not exhibit any specificity for molecular species of long chain diacylglycerols.

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

“…1, a and b), which in AtDGK2 includes amino acid residues 342-471. DGKc domains contain a presumed ATP binding site with a GXGXXG consensus sequence (where G represents glycine, and X represents any amino acid) (28). Although the presumed ATP binding site in AtDGK2 (Gly 401 -Ala 406 ) has an alanine instead of a glycine residue at the end of this motif, it is obvious from our results (see below) that this change does not render the enzyme inactive.…”

“…Interestingly, 1,2-SAG and 1,2-DOG are more unsaturated (18:0/20:4 and 18:1/18:1 for 1,2-SAG and 1,2-DOG, respectively) than the other lipids tested. Similarly, in other studies with human DGKs, a strong preference for 1,2-SAG and 1,2-DOG was observed (28,36). Saturated diacylglycerols (1,2-DOCG, 1,2-DPG, and 1,2-DMG) or monosaturated DAG analogs (1,2-OAG) were poor substrates for AtDGK2 (Fig.…”

Purification and characterization of sn-1-stearoyl-2-arachidonoylglycerol kinase from pig testes

“…The hDGKζ gene encoded for a protein of approx. 103 kDa that showed no selectivity for naturally occurring long-chain diradylglycerols [39]. However, analysis of the hDGKε cDNA showed that this sequence encoded for a 64 kDa protein that contained a DAG-binding site distinct from that found in the other DAG kinases.…”

“…The DGKδ gene was identified from a testis library and encodes a 130 kDa particulate protein that showed no DAG selectivity [37]. The other new DGK genes were isolated from human endothelial cells and were termed hDGKζ and hDGKε [38,39]. The hDGKζ gene encoded for a protein of approx.…”

Synthesis and Phorbol Ester Binding of the Cysteine-rich Domains of Diacylglycerol Kinase (DGK) Isozymes

“…To date, nine subtypes of mammalian DGKs have been cloned (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). All DGK isozymes consist of a conserved catalytic domain and two or three cysteine-rich C1 domains designated as C1A, C1B, and C1C (16).…”