Molecular cloning of a novel human diacylglycerol kinase highly selective for arachidonate-containing substrates.

Diacylglycerol (DAG) is a second messenger that activates protein kinase C and also occupies a central role in phospholipid biosynthesis. Conversion of DAG to phosphatidic acid by DAG kinase regulates the amount of DAG and the route it takes. We used degenerate primers to amplify polymerase chain reaction products from cDNA derived from human endothelial cells. A product with a novel sequence was identified and used to clone a 2.6-kilobase cDNA from an endothelial cell library. When transfected with a truncated version of this cDNA, COS-7 cells had a marked increase in DAG kinase activity, which demonstrated clear selectivity for arachidonoyl-containing species of diacylglycerol. The open reading frame of this clone has 567 residues with a predicted protein of 64 kDa. This enzyme, which we designated DGK epsilon, has two distinctive zinc finger-like structures in its N-terminal region, but does not contain the E-F hand motifs found in several other mammalian DGKs. The catalytic domain of DGK epsilon, which is related to other DGKs, contains two ATP-binding motifs. Northern blotting demonstrated that DGK epsilon is expressed predominantly in testis. This unique diacylglycerol kinase may terminate signals transmitted through arachidonoyl-DAG or may contribute to the synthesis of phospholipids with defined fatty acid composition.

Diacylglycerol occupies a central position in the biosynthesis of phospholipids and triglycerides. It also is an important intracellular messenger because it can bind to and activate protein kinase C, which, in turn, phosphorylates target proteins (1). This pathway has been implicated in many cellular response including growth, differentiation, and other events such as secretion. The mechanisms by which the signaling pathway and the synthesis of complex lipids are differentially regulated is not clear, but the concentration of DAG 1 within the cell is almost certainly one important component. In response to a variety of signals, the DAG level rises by the activation of one or more phospholipases C and, in some cases, a phospholipase D followed by phosphatidic acid phosphohydrolase. Either pathway causes a rise in the amount of diacylglycerol by degrading phospholipids. The level of DAG also is influenced by the rate at which it is converted into other products. One pathway for decreasing DAG is its conversion to phosphatidic acid, a reaction catalyzed by DAG kinases (EC 2.7.1.107).
The stimulated rise in DAG levels is an integral component of the response of cells to a variety of stimuli that lead to growth or differentiation, and the effects of phorbol esters, which are tumor promoters, are through activation of protein kinase C. Thus, the level of DAG may be an important determinant of growth. In support of this, we found that rapidly growing endothelial cells have severalfold higher levels of DAG than quiescent cells, and others observed that transformation of cells by several oncogenes results in an increased content of DAG even in the absence of an additional stimulus (2)(3)(4). The conversion of DAG to phosphatidic acid may dampen such signals, but the precise effects on cellular behavior are hard to predict because phosphatidic acid also may influence the growth response (5)(6)(7). Another role of the DAG kinase reaction may be to resynthesize phosphatidylinositol, which, unlike most phospholipids, has a characteristic fatty acid composition: 1-stearoyl-2-arachidonoyl (8). The mechanism for achieving this composition has never been elucidated although one possibility is that an enzyme(s) in the synthetic, or a salvage, pathway are specific for precursors with the appropriate molecular composition. Following the stimulated turnover of phosphatidylinositol, there is a later rise in phosphatidic acid, which has been thought to be the result of a DGK-catalyzed reaction. If this enzyme were specific for arachidonate-containing species of DAG, then multiple cycles might progressively enrich phosphatidylinositol with arachidonate.
The first isoform of DAG kinase characterized at a molecular level, DGK␣, has a molecular mass of about 80 kDa (9, 10) and is found predominantly in lymphocytes and oligodendrocytes (11). A second form, DGK␤, was cloned from brain where it is mainly expressed (12). Kai et al. (13) isolated a cDNA for DGK␥ from a human liver library, but subsequently found it to be expressed mostly in retina (13). A homologous rat cDNA is highly expressed in cerebellar cells (14). We recently identified DGK from human endothelial cells and showed that it has broad distribution, with highest levels in brain and muscle. 2 None of these isoforms exhibits a strong preference for substrates with specific fatty acids. However, MacDonald et al. (15,16) described an activity that had marked preference for DAG species that contain an arachidonoyl residue. Walsh et al. (17) reported the purification of DAG kinase from bovine testis and * This work was supported by National Institutes of Health Grant CA59548 and by Special Center of Research in ARDS Grant HL50153. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) U49379.
found it to have a molecular mass of 58 kDa. This activity was highest in testis, followed by brain and spleen. The purified enzyme showed a marked preference (up to 20-fold) for 1,2-DAG with arachidonate at the sn-2 position.
In the experiments reported here, we discovered a novel isoform of DAG kinase by molecular cloning of a cDNA from an endothelial cell library. When expressed in mammalian cells, it gives an enzyme that is specific for arachidonate-containing DAG. This enzyme, which we have named DGK⑀, may have an important role in signaling by regulating the concentration of DAG.
Reverse Transcription-PCR-Degenerate primers containing inosine were designed based on the amino acid sequences conserved among the sequenced catalytic domains of DGK isozymes. The forward primer, 5Ј-TG(C/T)GGIGGIGA(C/T)GGIACIGT(N)GG-3Ј (GP-1) where I is inosine, was based on the amino acid sequence, CGGDGTVG, and corresponded to the amino acids Cys 432 -Gly 439 of human DGK␣ (10) and the corresponding sequences, following optimal alignment, of other DGKs. The reverse primer, 5Ј-CAIGG(C/T)TCI(A/C)C(A/G)TCIA(C/T)(C/T)TG-CAT-3Ј (GP-2), was based on the amino acid sequence, MQ V/IDG/ VEPW, which corresponds to amino acids Met 695 -Trp 702 of human DGK␣ (10) and the aligned sequences of other DGKs except Drosophila DGK2 (19). Total RNA from confluent HUVEC monolayers was isolated by the guanidinium thiocyanate method (20). Single strand cDNA was synthesized with 1 g of total RNA from HUVEC at 37°C for 1 h using oligo(dT) 15 as the primer and M-MuLV reverse transcriptase (Life Technologies, Inc.). The reverse transcription mixture was then used as the template in the PCR amplification which was performed as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 75 s, 53°C for 2 min, and 72°C for 2 min, and concluding with a 5-min incubation at 72°C. The amplified PCR fragment (about 750 bp) was gel-purified and subcloned into pBluescript II SK(ϩ) (Stratagene) that had been cut with EcoRV and had a single T added (T-vector) (21). The cloned fragments were subjected to sequence analysis by the dideoxy chain termination method using a Sequenase 2.0 kit (U. S. Biochemical Corp.).
Screening of HUVEC cDNA Library-The cloned DGK fragment with a novel sequence was labeled with [␣-32 P]dCTP by PCR and used as a probe to screen a HUVEC gt11 cDNA library kindly provided by Dr. Evan Sadler (Washington University, St. Louis). The plaques were transferred to Hybond-N nylon membrane (Amersham) and hybridized overnight at 65°C in a solution containing 5 ϫ Denhardt's, 0.5 ϫ SSC, 0.1% SDS, and 0.1 mg/ml denatured herring sperm DNA. The filters were washed once in 2 ϫ SSC, 0.1% SDS, and once in 0.1 ϫ SSC, 0.1% SDS at room temperature for 20 min. Approximately 4 ϫ 10 6 plaques were screened. Positive clones were subcloned into pBluescript II SK(ϩ) and sequenced on both strands by the automated dideoxy method (ABI, University of Utah Cancer Center Core Sequencing Facility). COS-7 Transfection and Assay of DGK Activity-COS-7 transfection was carried out using LipofectAMINE reagent (Life Technologies, Inc.), and all steps were followed by the protocol provided by the manufacturer. After 2.5 days, the cells were harvested and lysed by sonication (twice with 5 watts for 5 s at 4°C) in the lysis buffer (0.1 ml/35 mm well) containing 20 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 4 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, a 20 g/ml concentration of each: leupeptin, pepstatin, aprotinin, and soybean trypsin inhibitor (13). The protein concentration of the cell homogenate was determined with the BCA protein assay reagent (Pierce). In two experiments, we also separated the homogenate into soluble and membrane-bound fractions by centrifugation at 100,000 ϫ g for 60 min (4°C). The pellet was resuspended in lysis buffer, and both fractions were assayed for DGK activity.
Since the full-length cDNA of DGK⑀ could not be expressed in COS-7 cells (discussed under "Results and Discussion"), a PCR product of the DGK⑀ cDNA lacking the 3Ј-untranslated region and part of the 5Јuntranslated region was amplified and cloned into pcDNAI/Neo (Invitrogen). The forward primer, 5Ј-GCATAAGCTCGATATCGAGG-TATCGTCCTTG-3Ј (GP-3) contained 10 random nucleotides, an EcoRV site (underlined), and 15 nucleotides complementary to nucleotide Ϫ21 to Ϫ7 of DGK⑀. The reverse primer, 5Ј-TTGTCTCGAGGTCGACATC-TATTCAGTCGCC-3Ј (GP-4) contained 10 random nucleotides, a SalI site (underlined), and 15 nucleotides complementary to nucleotide 1692-1706 of DGK⑀. The PCR amplification was performed as follows: 94°C for 30 s, 50°C for 30 s, and 72°C for 2 min and 30 s for 5 cycles, followed by 94°C for 30 s, 65°C for 30 s, and 72°C for 2 min and 30 s for 30 cycles using Pfu (Stratagene) DNA polymerase. The PCR product was gel-purified, cut with EcoRV/SalI, and cloned into the EcoRV/XhoI site of pcDNAI/Neo (Invitrogen).
The octyl glucoside/PS mixed-micelle assay of DGK activity was performed as described (17). In brief, the assay mixture contained 50 mM MOPS, pH 7.2, 100 mM NaCl, 20 mM MgCl 2 , 1 mM EGTA, 1 mM dithiothreitol, 2 mM diacylglycerol, 3.5 mM phosphatidylserine, 75 mM octyl-␤-glucopyranoside, 500 M [␥-32 P] ATP, and 20 g of cellular protein in a volume of 200 l. The reaction was initiated by the addition of [␥-32 P] ATP (20 -30 Ci/mol) and processed for 10 min at 24°C. To stop the reaction, we added 1 ml of MeOH, 1 ml of CHCl 3 , 0.7 ml of 1% perchloric acid, and 50 g of phosphatidic acid as carrier. The lower phase of each sample was washed twice with 2 ml of 1% perchloric acid and then dried. They were resuspended in 100 l of 9:1 CHCl 3 /MeOH and separated by thin layer chromatography (20 ϫ 20 cm Silica 60A plates; Whatman). The plates were developed in (325:75:25) CHCl 3 / MeOH/HOAc. The region containing phosphatidic acid was scraped, and the radioactivity was estimated by liquid scintillation spectrometry. The amount of phosphatidic acid formed per reaction was calculated by dividing the radioactivity in phosphatidic acid by the specific radioactivity of the ATP in the reaction.
Multiple Tissue Northern Blotting-Multiple tissue Northern blots were purchased from Clontech. The ApaI/SalI fragment corresponding to nucleotides 1582-2470 of DGK⑀ was gel-purified and labeled with [␣-32 P]dCTP by random priming. Hybridization was carried out in QuikHyb (Stratagene). The conditions of hybridization and washing were as described in the protocol provided by the manufacturer.

RESULTS AND DISCUSSION
cDNA Cloning of DGK⑀-In previous experiments we had observed increased DGK activity as endothelial cells became quiescent (22). To identify the enzyme(s) responsible for the increased activity, we carried out reverse transcription-PCR using degenerate primers and total RNA from confluent cultures of human endothelial cells. We cloned and sequenced 29 PCR products; 20 were identical with human DGK␣ (10). However, we also found two novel DGKs; they contained the conserved sequences that the PCR strategy was designed to detect, but otherwise had unique primary structure. One of the PCR products, which had a length of 761 bp, was named DGK and will be reported separately. 2 The other novel product had a length of 746 bp, and we designated it DGK⑀. This PCR fragment was used as a probe to screen a cDNA library from human endothelial cells. Of two million plaques screened, there was one positive, DGKE1 (Fig. 1A). The 5Ј-portion of DGKE1 was subsequently used as the probe in another round of screening, which yielded two more positive clones (out of 2 ϫ 10 6 ), DGKE2 and DGKE3 (Fig. 1A), all of which were sequenced.
The cDNA of DGK⑀ has an open reading frame encoding 567 amino acids including the initiator methionine (calculated M r ϭ 63,884) (Fig. 1B). The translation initiation codon corresponds well with the Kozak sequence (23). However, in the clones shown, we did not detect in-frame stop codons in the 5Ј-untranslated region, nor were there typical polyadenylation signals in the 3Ј-untranslated region. Thus, the full-length messenger RNA for this enzyme is likely to be larger than the clone we isolated (see below). In a subsequent experiment, we screened a library from human testis (Clontech) and isolated another DGK⑀ clone with a longer 5Ј region, and an in-frame stop codon was found at position Ϫ129 from the initiating methionine (data not shown). DGK⑀ has 34%, 36%, 36%, and 32% identity with human DGK␣ (10), rat DGK␤ (12), human DGK␥ (13), and human DGK, 2 respectively. However, DGK⑀ clearly differs from the other cloned DGKs as it does not contain the N-terminal conserved region and E-F hand sequences found in other mammalian DGKs (Fig. 2). Moreover, the two zinc finger-like cysteine-rich sequences (residues His 60 -Cys 108 and His 125 -Cys 177 , Fig. 1B) found in DGK⑀ have distinctive patterns; the sequence of the first is Cys-X 2 -Cys-X 9 -Cys-X 2 -Cys-X 7 -Cys-X 9 -Cys, while the second has the sequence: Cys-X 2 -Cys-X 14 -Cys-X 2 -Cys-X 7 -Cys-X 9 -Cys. These precise zinc finger motifs are not found in any other DGKs, or in protein kinases C. In particular, the number of amino acids separating the last two cysteines in both the first and the second zinc finger-like motifs is 9 in DGK⑀ instead of 5-8 in most other mammalian DGKs or protein kinases C (the other novel DGK that we identified, 2 DGK, also is unusual in that it has 9 and 10 amino acids separating the two final cysteines in the two motifs). These features make DGK⑀ unique among the known DGKs. However, the catalytic domain, which is in the C-terminal region of DGK⑀, contains two putative ATP binding motifs (Fig.  1B) and is moderately conserved: the percent identity of amino acids in this region is 41%, 42%, 42%, and 38% with human DGK␣ (10), rat DGK␤ (12), human DGK␥ (13), and human DGK, 2 respectively.
Characterization of DGK⑀-We subcloned the full-length cDNA of DGK⑀ into the XhoI site of pcDNAI/Neo and trans-fected it into COS-7 cells. In our initial experiments, we could not detect DGK activity after transient expression (data not shown). We considered the possibility that this result was caused by rapid degradation of the mRNA since the 3Ј-untrans- lated region of DGK⑀ contains A/T-rich sequences, which have been shown to confer instability. To examine this, we deleted the entire 3Ј-untranslated region and part of the 5Ј-untranslated region, cloned it into pcDNAI/Neo, and transfected it into COS-7 cells. With this construct, we obtained a marked increase in DGK activity compared to cells transfected with vector alone (Fig. 3A). In addition, the activity in the DGK⑀transfected cells was highly selective for 1-stearoyl-2arachidonoyl-sn-glycerol as compared to other DAGs. However, the activity and substrate preference of DAG kinases can depend greatly on the type of assay system chosen (17). To exclude the possibility that the substrate preference of this isoform was an artifact of the assay system, we compared the utilization of different molecular species of DAG in the same assay but with different isoforms: the E. coli DGK, DGK⑀, and DGK. As shown in Fig. 3B, only DGK⑀ was selective for arachidonoyl-DAG, which confirms that the substrate preference is an intrinsic property of this isoform. We also tested the effects of phosphatidylserine on the activity of DGK⑀ as it has been shown variously to activate (16,24) or inhibit (17). Our assay contained phosphatidylserine, so we cannot make a direct comparison with previous results, but the inclusion of an additional 3.5 mM phosphatidylserine only slightly increased the activity of DGK⑀ (data not shown). We also tested whether two compounds, R59022 and R59949, that inhibit some, but not all, isoforms of DGK (24), had any effect on DGK⑀. The activity in homogenates from transfected cells was inhibited by approximately 11% and 41% in the presence of 100 M R59022 or R59949, respectively. Thus, DGK⑀ does not seem to be a primary target for these inhibitors. We also found that the majority (83%) of the DGK⑀ activity was in the supernatant fraction when the homogenate was separated by centrifugation (data not shown). This may indicate that this is a different enzyme than that studied by Walsh et al. (17), as theirs appeared to be a membrane protein. However, our result should be interpreted cautiously as the recovery of enzymatic activity in the supernatant ϩ pellet fractions was only 42% of the total in the homogenate. The basis for this loss is not clear since a sample of DGK handled identically gave complete (Ͼ99%) recovery.
Tissue Distribution of DGK⑀-We screened multiple human tissues by Northern blotting and found that DGK⑀ is expressed in testis, at a lower level in ovary, and at a barely detectable level in skeletal muscle and pancreas (Fig. 4). The mRNA band of DGK detected in Northern blotting (about 8 kb, Fig. 4) is much larger than the cDNA we cloned (2557 bp), which suggests that the 5Ј-untranslated and/or the 3Ј-untranslated region(s) of the messenger RNA is (are) very long. CONCLUSION The enzyme encoded by the cDNA that we isolated, DGK⑀, is distinctive compared to other known DGKs as it is very selective for arachidonoyl-DAG. The basis for this property is not clear. The distinctive zinc finger-like structures, which likely are the sites for DAG binding, may contribute to the substrate specificity, but DGK, which does not have the arachidonoyl-DAG specificity, has similar sequences. A DAG kinase with such specificity might play an important role in phospholipid metabolism by producing a precursor of phosphatidylinositol, phosphatidic acid, that is enriched in arachidonic acid. Multi- FIG. 3. Diacylglycerol kinase ⑀ has marked specificity for arachidonate-containing diacylglycerols. COS-7 cells were transfected with vector alone (pcDNAI) or with a vector containing the coding sequence of DGK⑀ (pcDNAI/DGK⑀). Total lysates of the cells were assayed for diacylglycerol kinase activity as described under "Experimental Procedures." A, DGK activity was assayed with the following substrates (indicated on the right): 1,2-didecanoyl-sn-glycerol (10:0/10: 0), 1-palmitoyl-2-oleoyl-sn-glycerol (16:0/18:1), 1-oleoyl-2-palmitoyl-snglycerol (18:1/16:0), 1,2-dioleoyl-sn-glycerol (18:1/18:1), 1-stearoyl-2-oleoyl-sn-glycerol (18:0/18:1), 1-stearoyl-2-linoleoyl-sn-glycerol (18:0/18: 2), and 1-stearoyl-2-arachidonoyl-sn-glycerol (18:0/20:4). The data were collected from two independent transfections, which are shown as individual experiments. B, comparison of the substrate specificity of different diacylglycerol kinases. We carried out transfections as above, but, as a control, we also transfected cells with DGK (pcDNAI/DGK). Additionally, assays were performed with recombinant DGK from E. coli (1 l of enzyme from the Amersham diacylglycerol assay kit). The substrates used are indicated using the abbreviations as above. For ease of comparison, the values obtained with each DGK using 1-stearoyl-2-arachidonoyl-sn-glycerol (18:0/20:4) as the substrate are shown as 100%. The activities measured using the other substrates are expressed as relative to the arachidonate-containing diacylglycerol. The values shown are the averages of two separate experiments.
FIG. 4. Tissue-specific expression of DGK⑀: Northern blot analysis of mRNA from human tissues. Filters with poly(A ϩ ) RNA from multiple human tissues were purchased from Clontech and were hybridized with a 32 P-labeled 0.9-kb ApaI/SalI fragment of pBS/DGK⑀. Lanes: 1, heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, pancreas; 9, spleen; 10, thymus; 11, prostate; 12, testis; 13, ovary; 14, small intestine; 15, colon (mucosal lining); 16, peripheral blood leukocytes. ple cycles of phosphatidylinositol hydrolysis and resynthesis could lead to progressive enrichment in arachidonic acid. However, DGK⑀ seems unlikely to serve this function generally as it has a very restricted pattern of expression. However, it is possible that other tissues have very low level expression, below what we could detect by Northern blotting, since the arachidonate-specific DGK activity has been reported previously in aortic endothelium (25). Alternatively, there could be another isoform with similar substrate preference.
Walsh et al. (17) recently reported purification of an arachidonoyl-specific DGK from bovine testis, and it is likely that DGK⑀ is its homolog (17). For example, the bovine enzyme has an apparent M r of 58,000 which is comparable to the calculated M r of DGK⑀, they share the substrate specificity, and both are highest in testis. However, unlike the bovine enzyme, which is inhibited by PS and insensitive to DGK inhibitors, DGK⑀ is insensitive to PS and moderately inhibited by R59949. These differences may reflect the assay conditions used or may indicate that there are two isoforms of DGK with specificity for diacylglycerol substrates containing arachidonic acid.