Lipid species change for SV40 transformed fibroblasts from wild-type or from diacylglycerol kinase-ε (DGKε) or diacylglycerol kinase-α (DGKα) knock out mice, were determined for glycerophospholipids, poly-phosphatidylinositides (GPInsP n ), and diacylglycerol (DAG) using direct infusion mass spectrometry. Dramatic differences in arachidonate (20:4 fatty acid)-containing lipids were observed for multiple classes of glycerophospholipids and polyphosphatidylinositides between wild-type and DGKε knock out cells. However, no difference was observed in either the amount or the acyl chain composition of DAG between DGKε knock out and wild-type cells, suggesting that DGKε catalyzed the phosphorylation of a minor fraction of the DAG in these cells. The differences in arachidonate content between the two cell lines were greatest for the GPInsP n lipids and least for DAG. These findings indicate that DGKε plays a significant role in determining the enrichment of GPInsP n with 20:4 and that there is a pathway for the selective translocation of arachidonoyl-phosphatidic acid from the plasma membrane to the endoplasmic reticulum. In contrast, no substantial difference was observed in the acyl chain composition of any class of glycerophospholipid or diacylglycerol between lipid extracts from fibroblasts from wild-type mice or from DGKα knock out mice. However, the cells from the DGKα knock out mice had a higher concentration of DAG, consistent with the lack of down regulation of the major fraction of DAG by DGKα, in contrast with DGKε that is primarily responsible for enrichment of GPInsP n with arachidonoyl acyl chains.Eukaryotic diacylglycerol kinases (DGKs) 1 are a family of 10 known isoforms playing important roles in signal transduction (1-3). In this work we compare the properties of two isoforms of DGK, DGKα and DGKε. Although these two isoforms catalyze the same reaction, † This work was supported in part by a grant from the Natural Sciences and Engineering Research Council of Canada, grant 9848 (to R.M.E.) and from the National Institutes of Health Grants R01-CA95463 (to M.K.T.) and U54 GM069338 (to H.A.B.). they have very different modes of interaction with membranes and different mechanism of regulation. DGKα contains two E-F hands and is a Ca 2+ -dependent enzyme (4,5), although it can also exhibit Ca 2+ -independent activity when binding to membranes rich in phosphatidylethanolamine (GPEtn) or cholesterol (6). DGKα is an amphitropic enzyme that binds to membranes only upon activation (7,8). In contrast DGKε is the only isoform with a hydrophobic segment that promotes attachment of the protein to membranes (9). In addition, DGKε is also unique in being the only isoform of DGK that has specificity for DAG containing arachidonoyl chains (10,11) as well as other polyunsaturated acyl chains (12). Hence, the DAG derived from GPIns(4,5)P 2 hydrolysis by GPIns(4,5)P 2 -specific phospholipase C (PLC) will also be arachidonoyl-rich as will the phosphatidic acid (GPA) formed as a product of DGKε-catalyzed phosphoryla...

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