MOLECULAR-CLONING OF A NOVEL HUMAN DIACYLGLYCEROL KINASE HIGHLY SELECTIVE FOR ARACHIDONATE-CONTAINING SUBSTRATES

Diacylglycerol (DAG) is a second messenger that activates protein kina se C and also occupies a central role in phospholipid biosynthesis. Co nversion of DAG to phosphatidic acid by DAG kinase regulates the amoun t of DAG and the route it takes. We used degenerate primers to amplify polymerase chain reaction products from cDNA derived from human endot helial cells. A product with a novel sequence was identified and used to clone a 2,6-kilobase cDNA from an endothelial cell library. When tr ansfected with a truncated version of this cDNA, COS-7 cells had a mar ked increase in DAG kinase activity, which demonstrated clear selectiv ity for arachidonoyl-containing species of diacylglycerol. The open re ading frame of this clone has 567 residues with a predicted protein of 64 kDa. This enzyme, which we designated DGK epsilon, has two distinc tive zinc finger-like structures in its N-terminal region, but does no t contain the E-F hand motifs found in several other mammalian DGKs. T he catalytic domain of DGK epsilon, which is related to other DGKs, co ntains two ATP-binding motifs. Northern blotting demonstrated that DGK epsilon is expressed predominantly in testis. This unique diacylglyce rol kinase may terminate signals transmitted through arachidonoyl-DAG or may contribute to the synthesis of phospholipids with defined fatty acid composition.