Molecular cloning of a novel human diacylglycerol kinase highly selective for arachidonate-containing substrates.
Diacylglycerol (DAG) is a second messenger that activates protein kinase C and also occupies a central role in phospholipid biosynthesis. Conversion of DAG to phosphatidic acid by DAG kinase regulates the amount of DAG and the route it takes. We used degenerate primers to amplify polymerase chain reaction products from cDNA derived from human endothelial cells. A product with a novel sequence was identified and used to clone a 2.6-kilobase cDNA from an endothelial cell library. When transfected with a truncated version of this cDNA, COS-7 cells had a marked increase in DAG kinase activity, which demonstrated clear selectivity for arachidonoyl-containing species of diacylglycerol. The open reading frame of this clone has 567 residues with a predicted protein of 64 kDa. This enzyme, which we designated DGK epsilon, has two distinctive zinc finger-like structures in its N-terminal region, but does not contain the E-F hand motifs found in several other mammalian DGKs. The catalytic domain of DGK epsilon, which is related to other DGKs, contains two ATP-binding motifs. Northern blotting demonstrated that DGK epsilon is expressed predominantly in testis. This unique diacylglycerol kinase may terminate signals transmitted through arachidonoyl-DAG or may contribute to the synthesis of phospholipids with defined fatty acid composition.
Tang
W
W
Program in Human Molecular Biology & Genetics, the Nora Eccles Harrison Cardiovascular Research & Training Institute, University of Utah, Salt Lake City, Utah 84112.
Bunting
M
M
Zimmerman
G A
GA
McIntyre
T M
TM
Prescott
S M
SM
D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D019852:Diacylglycerol Kinase / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D013379:Substrate Specificity; D014018:Tissue Distribution
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D016718:Arachidonic Acid; D017853:Phosphotransferases (Alcohol Group Acceptor); C526464:DGKE protein, human; D019852:Diacylglycerol Kinase