Abstract
Diacylglycerol (DAG) occupies a central position in the synthesis of complex lipids and also has important signaling roles. For example, DAG is an allosteric regulator of protein kinase C, and the cellular levels of DAG may influence a variety of processes including growth and differentiation. 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 (Whatley, 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 (hDGKzeta) with a predicted molecular mass of 103.9 kDa. Human DGK zeta 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 hDGKzeta mRNA is expressed at highest levels in brain. COS-7 cells transfected with the hDGKzeta cDNA express 117-kDa and 114-kDa proteins that react specifically with an antibody to a peptide derived 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 activity. The hDGKzeta 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.
Highlights
Diacylglycerol (DAG) occupies a central position in the synthesis of complex lipids and has important signaling roles
The protein most closely related to hDGK is Drosophila DGK2, which is the product of the rdgA locus (Fig. 1B); the sequences of the two proteins are 42% identical and 61% similar
The Drosophila protein contains a much larger coding region due to a predicted extension of the amino terminus and does not include a key sequence found in hDGK that is potentially important for regulation
Summary
Materials—[␥-32P]ATP (6000 Ci/mmol), [␣-32P]dCTP (6000 Ci/mmol), ECL detection reagent, and Hybond-N were purchased from Amersham. The -actin probed Human Multiple Tissue Northern blot was washed at room temperature twice in 2 ϫ SSC, 0.1% SDS for 15 min followed by two washes in 0.1 ϫ SSC, 0.1% SDS for 15 min prior to immunological detection. Each membrane was incubated with the anti-peptide antibody (1:1000 dilution) in 20 ml of TBST with 5% nonfat dry milk for 1 h. The membranes were incubated for 1 h with a goat F(abЈ) anti-rabbit immunoglobulin antibody at a 1:2000 dilution in TBST with 5% nonfat dry milk. This secondary antibody had been affinity-isolated, preadsorbed with human immunoglobulin, and conjugated with horseradish peroxidase. The membranes were developed with the ECL detection reagent (Amersham) according to manufacturer specification
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