Publications

• Diacylglycerol kinase epsilon is selective for both acyl chains of phosphatidic acid or diacylglycerol.

  Lung M., Shulga Y.V., Ivanova P.T., Myers D.S., Milne S.B., Brown H.A., Topham M.K., Epand R.M.

  The phosphatidylinositol (PI) cycle mediates many cellular events by controlling the metabolism of many lipid second messengers. Diacylglycerol kinase epsilon (DGK epsilon) has an important role in this cycle. DGK epsilon is the only DGK isoform to show inhibition by its product phosphatidic acid ... >> More

  The phosphatidylinositol (PI) cycle mediates many cellular events by controlling the metabolism of many lipid second messengers. Diacylglycerol kinase epsilon (DGK epsilon) has an important role in this cycle. DGK epsilon is the only DGK isoform to show inhibition by its product phosphatidic acid (PA) as well as substrate specificity for sn-2 arachidonoyl-diacylglycerol (DAG). Here, we show that this inhibition and substrate specificity are both determined by selectivity for a combination of the sn-1 and sn-2 acyl chains of PA or DAG, respectively, preferring the most prevalent acyl chain composition of lipids involved specifically in the PI cycle, 1-stearoyl-2-arachidonoyl. Although the difference in rate for closely related lipid species is small, there is a significant enrichment of 1-stearoyl-2-arachidonoyl PI because of the cyclical nature of PI turnover. We also show that the inhibition of DGK epsilon by PA is competitive and that the deletion of the hydrophobic segment and cationic cluster of DGK epsilon does not affect its selectivity for the acyl chains of PA or DAG. Thus, this active site not only recognizes the lipid headgroup but also a combination of the two acyl chains in PA or DAG. We propose a mechanism of DGK epsilon regulation where its dual acyl chain selectivity is used to negatively regulate its enzymatic activity in a manner that ensures DGK epsilon remains committed to the PI turnover cycle. This novel mechanism of enzyme regulation within a signaling pathway could serve as a template for the regulation of enzymes in other pathways in the cell. << Less

  J. Biol. Chem. 284:31062-31073(2009) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.

• Study of arachidonoyl specificity in two enzymes of the PI cycle.

  Shulga Y.V., Topham M.K., Epand R.M.

  We identified a conserved pattern of residues L-X((3-4))-R-X((2))-L-X((4))-G, in which -X((n))-is n residues of any amino acid, in two enzymes acting on the polyunsaturated fatty acids, diacylglycerol kinase epsilon (DGKɛ) and phosphatidylinositol-4-phosphate-5-kinase Iα (PIP5K Iα). DGKɛ is the on ... >> More

  We identified a conserved pattern of residues L-X((3-4))-R-X((2))-L-X((4))-G, in which -X((n))-is n residues of any amino acid, in two enzymes acting on the polyunsaturated fatty acids, diacylglycerol kinase epsilon (DGKɛ) and phosphatidylinositol-4-phosphate-5-kinase Iα (PIP5K Iα). DGKɛ is the only one of the 10 mammalian isoforms of DGK that exhibits arachidonoyl specificity and is the only isoform with the motif mentioned above. Mutations of the essential residues in this motif result in the loss of arachidonoyl specificity. Furthermore, DGKα can be converted to an enzyme having this motif by substituting only one residue. When DGKα was mutated so that it gained the motif, the enzyme also gained some specificity for arachidonoyl-containing diacylglycerol. This motif is present also in an isoform of phosphatidylinositol-4-phosphate-5-kinase that we demonstrated had arachidonoyl specificity for its substrate. Single residue mutations within the identified motif of this isoform result in the loss of activity against an arachidonoyl substrate. The importance of acyl chain specificity for the phosphatidic acid activation of phosphatidylinositol-4-phosphate-5-kinase is also shown. We demonstrate that the acyl chain dependence of this phosphatidic acid activation is dependent on the substrate. This is the first demonstration of a motif that endows specificity for an acyl chain in enzymes DGKε and PIP5K Iα. << Less

  J. Mol. Biol. 409:101-112(2011) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Molecular cloning of a diacylglycerol kinase isozyme predominantly expressed in human retina with a truncated and inactive enzyme expression in most other human cells.

  Kai M., Sakane F., Imai S., Wada I., Kanoh H.

  In order to clone novel diacylglycerol kinase (DGK) isozymes, we first obtained a DGK-related cDNA fragment by polymerase chain reaction using the human hepatoma cell line HepG2 mRNA and degenerated primers. The amplified fragment was subsequently used as a probe for screening the cDNA library fro ... >> More

  In order to clone novel diacylglycerol kinase (DGK) isozymes, we first obtained a DGK-related cDNA fragment by polymerase chain reaction using the human hepatoma cell line HepG2 mRNA and degenerated primers. The amplified fragment was subsequently used as a probe for screening the cDNA library from HepG2 cells. We obtained a cDNA clone coding for a novel DGK isozyme (designated DGK gamma) comprised of 791 amino acid residues. The amino acid sequence of DGK gamma was 52 and 62% identical to those of previously sequenced porcine 80-kDa and rat 90-kDa enzymes, respectively. DGK gamma, although initially cloned from the HepG2 cDNA libraries, was unexpectedly expressed in the human retina abundantly and to a much lesser extent in the brain. Other human tissues, including the liver and HepG2 cells, contained extremely low levels of DGK gamma mRNA. Furthermore, HepG2 cells and most of the human tissues except for the retina and brain expressed a truncated DGK gamma with an internal deletion of 25 amino acid residues (Ile451-Gly475). When transfected into COS-7 cells, the nontruncated cDNA gave phosphatidylserine-dependent DGK activity with no apparent specificity with regard to the acyl compositions of diacylglycerol. In contrast the truncated cDNA failed to give DGK activity in spite of the expression of its mRNA and enzyme protein in COS cells, thus demonstrating that the truncated DGK gamma is catalytically inactive. The sequence comparison of the three cloned DGKs revealed the presence of four highly conserved regions including the two sets each of EF-hand and zinc finger structures. Although the implication of the catalytically inactive form of DGK gamma remains unknown, this work further demonstrates the occurrence of multiple animal DGK isozymes with a conserved basic structure but with markedly different expression patterns depending on the cell types. << Less

  J. Biol. Chem. 269:18492-18498(1994) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Substrate chirality and specificity of diacylglycerol kinases and the multisubstrate lipid kinase.

  Epand R.M., Shulga Y.V., Timmons H.C., Perri A.L., Belani J.D., Perinpanathan K., Johnson-McIntire L.B., Bajjalieh S., Dicu A.O., Elias C., Rychnovsky S.D., Topham M.K.

  The alpha, zeta, and epsilon isoforms of diacylglycerol kinase exhibit a high degree of stereospecificity in the phosphorylation of diacylglycerol. In comparison, a multiple lipid kinase, MuLK, shows much less stereospecificity, phosphorylating 1,2-dioleoylglycerol only approximately 2-3 times mor ... >> More

  The alpha, zeta, and epsilon isoforms of diacylglycerol kinase exhibit a high degree of stereospecificity in the phosphorylation of diacylglycerol. In comparison, a multiple lipid kinase, MuLK, shows much less stereospecificity, phosphorylating 1,2-dioleoylglycerol only approximately 2-3 times more rapidly than 2,3-dioleoylglycerol. The alpha and zeta isoforms of diacylglycerol kinase are inhibited by 2,3-dioleoylglycerol, but not the more substrate-selective epsilon isoform. The inhibition by 2,3-dioleoylglycerol is uncompetitive. This corresponds to a kinetic scheme in which the inhibitor can bind to the enzyme-substrate complex, but not to the free enzyme. Our data indicate that despite their similar structures, 1,2-dioleoylglycerol and 2,3-dioleoylglycerol do not compete for the active site of these three isoforms of diacylglycerol kinase. We suggest that the 2,3-dioleoylglycerol binds to a site on the alpha and zeta isoforms of diacylglycerol kinase that is exposed as a consequence of the substrate binding to the active site. The chiral specificity of these enzymes thus mimics the substrate specificity, with MuLK being the least selective and the epsilon isoform of diacylglycerol kinase exhibiting the greatest selectivity. << Less

  Biochemistry 46:14225-14231(2007) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The cloning and characterization of a novel human diacylglycerol kinase, DGK-iota.

  Ding L., Traer E., McIntyre T.M., Zimmerman G.A., Prescott S.M.

  Diacylglycerol (DAG) plays a central role in both the synthesis of complex lipids and in intracellular signaling; diacylglycerol kinase (DGK) catalyzes the phosphorylation of DAG, which yields phosphatidic acid. A family of DGKs has been identified in multicellular organisms over the past few year ... >> More

  Diacylglycerol (DAG) plays a central role in both the synthesis of complex lipids and in intracellular signaling; diacylglycerol kinase (DGK) catalyzes the phosphorylation of DAG, which yields phosphatidic acid. A family of DGKs has been identified in multicellular organisms over the past few years, but the physiological function(s) of this diversity is not clear. One clue has come from the Drosophila DGK2, rdgA, since mutations in this gene cause retinal degeneration. We isolated a novel DGK, which we designated DGKiota, from human retina and brain libraries. DGKiota contains two cysteine-rich repeats, a region similar to the phosphorylation site domain of myristoylated alanine-rich C kinase substrate, a conserved catalytic domain, and four ankyrin repeats at its C terminus. By primary structure, it is most similar to human DGKzeta and Drosophila rdgA. An >12-kilobase mRNA for DGKiota was detected only in brain and retina among the tissues examined. In cells transfected with the DGKiota cDNA, we detected an approximately 130-kDa protein by immunoassay, and activity assays demonstrated that it encodes a functional DAG kinase. The protein was found to be in both the cytoplasm and nucleus with the localization controlled by PKC isoforms alpha and gamma. The gene encoding DGKiota was localized to human chromosome 7q32.3-33, which is known to be a locus for an inherited form of retinitis pigmentosa. These results have defined a novel isoform of DAG kinase, which may have important cellular functions in the retina and brain. << Less

  J. Biol. Chem. 273:32746-32752(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Molecular cloning and characterization of a novel human diacylglycerol kinase zeta.

  Bunting M., Tang W., Zimmerman G.A., McIntyre T.M., Prescott S.M.

  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 ... >> More

  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. << Less

  J. Biol. Chem. 271:10230-10236(1996) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Alternative splicing of the human diacylglycerol kinase zeta gene in muscle.

  Ding L., Bunting M., Topham M.K., McIntyre T.M., Zimmerman G.A., Prescott S.M.

  Diacylglycerol can function as a second messenger, and one mechanism for the attenuation of this signal is its conversion to phosphatidic acid, which is catalyzed by diacylglycerol kinase (DGK). We screened a cDNA library from human skeletal muscle and isolated two DGKzeta cDNAs that differed from ... >> More

  Diacylglycerol can function as a second messenger, and one mechanism for the attenuation of this signal is its conversion to phosphatidic acid, which is catalyzed by diacylglycerol kinase (DGK). We screened a cDNA library from human skeletal muscle and isolated two DGKzeta cDNAs that differed from the 3.5-kb clone originally identified in endothelial cells. One transcript, which was 3.4 kb long, was shown to be nonfunctional; it had a 77-bp deletion that included the translation initiation site. The other was 4.1 kb long with a unique 5' sequence of 853 bp. We also isolated a genomic clone of DGKzeta and determined its organization and location; it contains 32 exons, spans approximately 50 kb of genomic sequence, and maps to chromosome 11p11.2. The protein encoded by the 4.1-kb transcript contains two cysteine-rich regions, a catalytic domain, and ankyrin repeats like the endothelial form of DGKzeta, as well as a unique N-terminal domain. The coding sequence was shown to be derived from alternative splicing of the DGKzeta gene. In cells transfected with the 4.1-kb clone, we detected a 130-kDa protein with an antibody to DGKzeta and demonstrated that it was localized predominantly in the nucleus. We conclude that alternative splicing generates tissue-specific variants of DGKzeta that share some properties but may have unique ones as well. << Less

  Proc. Natl. Acad. Sci. U.S.A. 94:5519-5524(1997) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.