Dramatic Differences in the Roles in Lipid Metabolism of Two Isoforms of Diacylglycerol Kinase

Abstract

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...

Diacylglycerol Kinase ϵ Is Selective for Both Acyl Chains of Phosphatidic Acid or Diacylglycerol

“…The PI cycle mediates a large variety of cellular events by controlling the metabolism of several important lipid second messengers, including several phosphoinositides (43), DAG and PA, each possessing their own diverse range of signaling roles in the cell. This postulation of DGK⑀ function has been supported by recent in vivo data in which we show that DGK⑀ KO mouse embryonic fibroblast (MEF) cells have a significant reduction in PI with arachidonoyl side chains compared with WT cells (44).…”

“…Mass spectral analysis was performed by direct infusion on a Finnigan TSQ Quantum triple-quadrupole mass spectrometer essentially as described previously (44). Identification of the individual phospholipids was accomplished by liquid chromatography-tandem mass spectrometry by utilization of Applied Biosystems/MDS SCIEX 4000 QTRAP hybrid triple quadrupole/linear ion trap mass spectrometer and a Shimadzu high pressure liquid chromatography system with a Phenomenex Luna Silica column (2 ϫ 250 mm, 5-m particle size) using a gradient elution as communicated elsewhere (44,53). The identification of the individual species was based on their chromatographic and mass spectral characteristic (44,53).…”

“…It is instructive to compare these lipodomics results (obtained from DGK⑀ KO MEF cells) (44) with an earlier study in which DGK⑀ was overexpressed in porcine aortic endothelial cells (45). Overexpression of DGK⑀ results in the selective removal of DAGs with certain acyl chains, including those with an arachidonoyl group (45).…”

“…Overexpression of DGK⑀ results in the selective removal of DAGs with certain acyl chains, including those with an arachidonoyl group (45). In contrast, there is no concentration or acyl chain difference among the different species of DAG when comparing WT with DGK⑀ KO MEFs (44). Thus, although one would anticipate an increase in arachidonoyl-containing DAG in the DGK⑀ KO MEF cells, because there are many pathways for the removal of DAG (including other isoforms of DGK), this difference is not observed.…”

The Roles of Diacylglycerol Kinases in the Central Nervous System: Review of Genetic Studies in Mice

“…Further, in DGKe KO mice, the phosphatidylinositol 4,5bisphosphatesignaling pathway in the cerebral cortex was greatly affected, leading to lower accumula tion of 20:4DAG and free 20:4, and seizures were decreased compared with wildtype mice (27). In normal conditions, DGKeKO mice had lower PI content with 1stearoyl2arachidonoyl acyl chains (68).…”