MOLECULAR-CLONING AND CHARACTERIZATION OF A NOVEL HUMAN DIACYLGLYCEROL KINASE-ZETA

Diacylglycerol (DAG) occupies a central position in the synthesis of c omplex Lipids and also has important signaling roles. For example, DAG is an allosteric regulator of protein kinase C, and the cellular leve ls of DAG may influence a variety of processes including growth and di fferentiation. We previously demonstrated that human endothelial cells derived from umbilical vein express growth-dependent changes in their basal levels of diacylglycerol and diacylglycerol kinase activity (Wh atley, R. E., Stroud, E. D., Bunting, M., Zimmerman, G. A., McIntyre, T. M., and Prescott, S. M. (1993) J. Biol. Chem. 268, 16130-16138). 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 zeta) with a predict ed molecular mass of 103.9 kDa. Human DGK zeta contains two zinc finge rs, an ATP binding site, and four ankyrin repeats near the carboxyl te rminus, A unique feature, as compared with other diacylglycerol kinase s, is the presence of a sequence homologous to the MARCKS phosphorylat ion site domain. From Northern blot analysis of multiple tissues, we o bserved that hDGK zeta mRNA is expressed at highest levels in brain, C OS-7 cells transfected with the hDGK zeta cDNA express 117-kDa and 114 -kDa proteins that react specifically with an antibody to a peptide de rived from a unique sequence in hDGK zeta. The transfected cells also express increased diacylglycerol kinase activity, which is not altered in the presence of R59949, an inhibitor of human platelet DGK activit y. The hDGK zeta displays stereoselectivity for 1,2-diacylglycerol spe cies in comparison to 1,3-diacylglycerol, but does not exhibit any spe cificity for molecular species of long chain diacylglycerols.