©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Cloning and Characterization of a Novel Human Diacylglycerol Kinase (*)

(Received for publication, November 22, 1995; and in revised form, February 22, 1996)

Michaeline Bunting Wen Tang Guy A. Zimmerman Thomas M. McIntyre Stephen M. Prescott (§)

From the Eccles Program in Human Molecular Biology & Genetics, the Nora Eccles Harrison Cardiovascular Research & Training Institute, and the Departments of Internal Medicine, Pathology, and Biochemistry, University of Utah, Salt Lake City, Utah 84112

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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 (hDGK) with a predicted molecular mass of 103.9 kDa. Human DGK 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 hDGK mRNA is expressed at highest levels in brain. COS-7 cells transfected with the hDGK cDNA express 117-kDa and 114-kDa proteins that react specifically with an antibody to a peptide derived from a unique sequence in hDGK. 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 hDGK 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.


INTRODUCTION

1,2-Diacylglycerol (DAG) (^1)occupies a central position in the biosynthesis of complex lipids and is a key intracellular messenger by virtue of its ability to activate protein kinase C (PKC). How DAG can serve two seemingly different, and essential, roles in cellular processes is not clear, but one important issue is likely to be the intracellular concentration at a given time. The intracellular DAG levels are regulated by the rates of both synthesis and degradation. Receptor-mediated activation of a phospholipase C, or phospholipase D followed by a phosphatidate phosphohydrolase, have been shown to increase intracellular DAG levels (1) . One potential fate of the DAG produced during these responses is to be phosphorylated in a reaction that uses ATP as a phosphate donor. This reaction is catalyzed by DAG kinase (DGK) (2) (EC 2.7.1.107) and yields phosphatidic acid. This enzymatic conversion is thought to be a key mechanism by which PKC activation is attenuated by virtue of lowering the intracellular concentration of DAG. However, phosphatidic acid has been shown to be mitogenic (3) and to modulate the activity of intracellular proteins including n-chimaerin (4) and NF-1(5) . Further, it too is a central metabolite in complex lipid synthesis. Thus, the conversion of DAG to phosphatidic acid may have complicated net effects.

DGK activities have been identified from a wide range of cellular sources. Further analysis of the activity from these tissues and cell types by protein purification, nucleic acid hybridization, and reverse transcription-PCR has identified several isoforms of DAG kinase. An 80-kDa isozyme, DGKalpha, was the first isoform purified and cloned from porcine (6) and human tissues(7) . This enzyme is primarily expressed in lymphocytes and oligodendrocytes(8) . A cDNA for a second DGK isozyme, DGKbeta, subsequently was isolated from a rat brain library and encodes a protein with a predicted mass of 90 kDa. DGKbeta is primarily expressed within adult brain cell populations including the olfactory tubercle, nucleus accumbens, and the caudate putamen(9) . Another form, DGK, was cloned from a human HepG2 library and found to be expressed predominantly in retina(10) . A hDGK cDNA containing a 25-amino acid deletion within the catalytic domain was identified, suggesting that this isoform may be regulated by alternative splicing. Recently, a rat DGK cDNA displaying significant homology to hDGK was isolated and found to be highly expressed in cerebellar Purkinje cells (11) . All of these isoforms have a similar domain structure including two E-F hands, two zinc fingers, and a catalytic domain containing a predicted ATP binding site. Additional DGK activities have been purified from a variety of sources. Some of these kinases display unique characteristics that differ from DGKalpha, -beta, and -, suggesting the presence of additional DGK isozymes. For example, a unique 58-kDa DGK, which preferentially phosphorylates DAG species containing arachidonate, recently was purified to homogeneity from bovine testis(12) . Finally, several homologous cDNAs have been identified in Drosophila(13, 14, 15) . Although the enzymatic activities of the corresponding proteins have not been well characterized, they represent another set of potentially important kinases.

Intracellular DAG levels have been shown to play a vital role in cellular growth responses. Moreover, the tumor-promoting effects of phorbol esters are thought to derive from their ability to activate protein kinase C isozymes, and, therefore, an increased intracellular concentration of DAG could be an endogenous tumor promoter. Oncogenic transformation by ras(16) , sis(16) , src(17) , fms(17) , and erbB(18) all have been demonstrated to elevate basal intracellular DAG. We showed that rapidly growing human endothelial cells maintain a 2- to 3-fold higher level of DAG than confluent cells and that PKC was activated in the dividing cells(19) . Inversely, both DAG kinase and DAG lipase (20) activities increased as the endothelial cells reached confluence suggesting that metabolism of DAG occurred through one or both enzymes. Interestingly, the translocation of DAG kinase from the cytosol to membranes observed in stimulated cells is diminished in ras- (21) , src-(22) , or erbB- (22) transformed fibroblasts. Further, the elevated DAG levels in v-Ki-ras-transformed NIH/3T3 cells can be lessened by overexpression of DGKalpha(23) . To evaluate more critically the role of DAG kinase in endothelial cell responses and growth control, we conducted experiments to identify the isozymes of DAG kinase expressed by human endothelial cells and, in the process, discovered a novel isoform, which we have named hDGK.


EXPERIMENTAL PROCEDURES

Materials

[-P]ATP (6000 Ci/mmol), [alpha-P]dCTP (6000 Ci/mmol), ECL detection reagent, and Hybond-N were purchased from Amersham. Polyvinylidene difluoride was purchased from Millipore. Octyl-beta-glucopyranoside (Ultrol grade) and R59949 were purchased from Calbiochem. Phosphatidylserine and phosphatidic acid were purchased from Avanti Polar Lipids (Alabaster, AL). All other lipid compounds were purchased from Serdary. Dulbecco's modified Eagle's medium, penicillin, streptomycin, Moloney murine leukemia virus reverse transcriptase, oligo(dT), and LipofectAMINE were purchased from Life Technologies, Inc. Fetal bovine serum was purchased from Hyclone Laboratories (Logan, UT). Digoxigenin labeling and detection kit, soybean trypsin inhibitor, leupeptin, pepstatin, and aprotinin were purchased from Boehringer Mannheim. ATP, phenylmethylsulfonyl fluoride, Hanks' balanced salt solution, Ponceau red, and trichloroacetic acid were purchased from Sigma. Sequenase was obtained from U. S. Biochemical Corp. The Human Multiple Tissue Northern blot was purchased from Clontech. The horseradish peroxidase-conjugated goat F(ab`)(2) anti-rabbit immunoglobulin antibody was purchased from Biosource International. Oligonucleotides were synthesized at the University of Utah peptide/DNA user facility.

Cell Culture and Platelet Isolation

Primary cultures of human umbilical vein endothelial cells (HUVEC) were plated and maintained as described(24) . COS-7 cells were cultured in Dulbecco's modified Eagle's medium with high glucose containing 110 mg/liter pyruvate, 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin sulfate. Human platelets were isolated from whole blood as described previously(25) .

cDNA Synthesis and Degenerate PCR

Total RNA was isolated from confluent HUVEC monolayers(26) , and cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase and oligo(dT) as a primer. Two degenerate PCR primers (5`-TG(C/T)GGIGGIGA(C/T)GGIACIGTNGG-3`) and (5`-CAIGG(C/T)TCI(A/C)C(A/G)TCIA(C/T)(C/T)TGCAT-3`) were used to amplify HUVEC cDNA as follows: 94 °C for 5 min, (94 °C for 75 s, 53 °C for 2 min, 72 °C for 2 min) 30 cycles, and 72 °C for 5 min. The resulting PCR product was gel-purified and directly cloned into the EcoRV site of pBluescript II (Stratagene). Single clones were sequenced by manual dideoxy sequencing (Sequenase, U. S. Biochemical Corp.).

cDNA Isolation and Sequencing

The unique 761-bp PCR product was labeled with [alpha-P]dCTP by random priming and used to screen two cDNA libraries: one from endothelial cells in the basal state (a gift from Evan Sadler, Washington University, St. Louis), and the other from TNF-stimulated endothelial cells (from Don Staunton, ICOS Corp., Bothell, WA). All colonies and plaques were transferred to Hybond-N nylon membranes (Amersham) according to manufacturer guidelines. Membranes were hybridized with the hDGK PCR probe at 65 °C for 12 to 18 h in a solution containing 5 times Denhardt's, 0.5 times SSC, 0.1% SDS, and 100 µg/ml denatured herring sperm DNA. The membranes were washed, following hybridization, for 20 min in 2 times SSC, 0.1% SDS and 20 min in 0.1 times SSC, 0.1% SDS at room temperature. All membranes were exposed to x-ray film for a minimum of 12 h prior to development. After screening 4 times 10^5 recombinants, 17 positive single phage clones were isolated from the unstimulated endothelial cell library. Additionally, 6 positive single clones were isolated from the TNF-stimulated endothelial cell library after screening 7.5 times 10^5 recombinants. Two of the largest clones from the former and all of the clones from the latter library were analyzed by a combination of restriction mapping and partial nucleic acid sequencing. All of the clones that were analyzed demonstrated similar restriction maps. The largest of the representative clones from the TNF-stimulated endothelial cell library was selected for further analysis and sequenced from both strands by a combination of automated (ABI, University of Utah Cancer Center Core Sequencing Facility) and manual dideoxy sequencing (Sequenase, U. S. Biochemical Corp.).

Northern Blot

RNA was isolated from confluent HUVEC monolayers as described previously(26) . Total RNA (10 µg/lane) was electrophoresed through a 0.8% agarose gel containing formaldehyde and blotted to a Hybond-N membrane by capillary transfer. Probe synthesis, hybridization, and detection methods for all Northern blots were performed according to Boehringer Mannheim specifications for digoxigenin-labeled probes. A pBluescript II vector containing the hDGK PCR-amplified 761-bp product was linearized with HindIII and used as a template for digoxigenin-labeled riboprobe synthesis. DGK-probed membranes were washed twice at room temperature in 2 times SSC with 0.1% SDS for 5 min followed by two washes in 0.1 times SSC, 0.1% SDS at 68 °C for 15 min followed by incubation for 1 h at 68 °C in 50% formamide, 50 mM Tris-HCl, pH 8.0, and 1% SDS prior to immunological detection. The beta-actin cDNA probe was purchased from Clontech and labeled with digoxigenin by random priming. Following hybridization, the beta-actin probed Human Multiple Tissue Northern blot was washed at room temperature twice in 2 times SSC, 0.1% SDS for 15 min followed by two washes in 0.1 times SSC, 0.1% SDS for 15 min prior to immunological detection.

COS-7 Transfection

The hDGK cDNA was expressed in pcDNA 1/AMP (Invitrogen) in the forward orientation. Each p35 dish containing COS-7 cells at 60-80% confluence was transfected with 1 µg of plasmid DNA and 5 µl of LipofectAMINE according to manufacturer specifications (Life Technologies Inc.). The cells were harvested by washing with ice-cold Hanks' balanced salt solution (Ca- and Mg-free) and were scraped into resuspension buffer (20 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 4 mM EGTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 20 µg/ml each leupeptin, pepstatin, aprotinin, and soybean trypsin inhibitor) (10) 48 h following transfection. All homogenates were frozen on dry ice and stored at -70 °C until assayed.

DAG Kinase Assay

Cell homogenates were sonicated twice with 5 watts for 5 s at 4 °C. The protein concentration of each cell homogenate was measured following trichloroacetic acid precipitation utilizing the BCA protein assay reagent (Pierce). Each assay contained 50 mM MOPS, pH 7.2, 100 mM NaCl, 20 mM MgCl(2), 1 mM EGTA, 1 mM dithiothreitol, 2 mM 1,2-dioleoyl-sn-glycerol, 3.5 mM phosphatidylserine, 75 mM octyl-beta-glucopyranoside, 500 µM [-P]ATP, and 0-20 µg of cell homogenate in a volume of 200 µl(12) . For substrate specificity experiments, various diacylglycerol species were substituted for 1,2-dioleoyl-sn-glycerol. The reactions were initiated by the addition of ATP (20-30 µCi/µmol), were incubated for 10 min at 24 °C, and were terminated by the addition of 200 µl of 1% perchloric acid. Then, to each tube, we added 1.0 ml of MeOH, 1.0 ml of CHCl(3), 500 µl of 1% perchloric acid, and 50 µg of phosphatidic acid, vortexed them well, and centrifuged them for 10 min at 400 times g. The lower phase of each sample was washed twice with 2 ml of 1% perchloric acid. The remaining lower phase was dried under N(2) and resuspended in 100 µl of 9:1 CHCl(3)/MeOH. Fifty µl of each sample were counted directly by liquid scintillation spectrometry, and the remainder was applied to a 20 times 20 cm Silica 60A thin layer chromatography plate (Whatman). The plates were developed in (325:75:25) CHCl(3)/MeOH/HOAc. The region containing phosphatidic acid (as indicated by radiolabeled standards) and the remaining areas of each lane were scraped separately, and the radioactivity was estimated by liquid scintillation spectrometry. The amount of phosphatidic acid formed per reaction was calculated as [2(direct count)(PA cpm)/(total lane cpm)] times [mole ATP/cpm]. R59949 was dissolved in dimethyl sulfoxide and added to relevant enzymatic reactions just prior to the addition of ATP.

Antibody Production and Western Blot Analysis

An anti-peptide rabbit polyclonal antibody was made by Quality Controlled Biochemicals (Hopkinton, MA) to the carboxyl-terminal peptide ((C)LENRQHYQMIQREDQE). This peptide corresponds to hDGK residues Leu to Glu with an additional N-terminal cysteine. The extra cysteine residue was used to couple the peptide to a carrier protein, keyhole limpet hemocyanin, prior to immunization. The antibody was isolated by precipitation with caprylic acid from rabbit serum and dialyzed overnight in phosphate-buffered saline at 4 °C. Samples from transfected cells (25 µg of protein) were loaded on a 7.5% acrylamide SDS-polyacrylamide gel electrophoresis gel and, following electrophoresis, were transferred to polyvinylidene difluoride. The lane corresponding to the molecular weight standards (Bio-Rad) was cut from the blot and stained with 0.4% Ponceau red, 0.3% trichloroacetic acid and destained in water. The primary antibody was diluted with an equal volume of either phosphate-buffered saline or 100 µg/ml peptide and incubated for 30 min at room temperature prior to incubation with the membrane. All subsequent incubation steps were performed at room temperature while shaking. Each membrane was first blocked in for 2 h in 100 ml of TBST (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 0.1% Tween 20) containing 5% nonfat dry milk. Each membrane was then incubated with the anti-peptide antibody (1:1000 dilution) in 20 ml of TBST with 5% nonfat dry milk for 1 h. The membranes subsequently were washed 5 times each for 10 min in 100 ml of TBST containing 5% nonfat dry milk. The membranes then were incubated for 1 h with a goat F(ab`)(2) 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 subsequently washed twice for 20 min, and then twice for 10 min, with 100 ml of TBST. The membranes were then developed with the ECL detection reagent (Amersham) according to manufacturer specification.


RESULTS

Cloning of a cDNA Encoding DGK

We used degenerate primers complementary to the conserved catalytic domain of known DAG kinases, and cDNA from human endothelial cells as a template, to amplify fragments of approximately 750 bp by PCR. This product was cloned directly into pBluescript II, and single clones were subjected to nucleic acid sequencing. Clones representing DGKalpha were represented, but we also observed a 761-bp clone that displayed similarity to known DAG kinases but was not identical with any of them. We designated this novel clone as human diacylglycerol kinase (hDGK). The unique PCR fragment was used to screen two cDNA libraries derived from human endothelial cells, and multiple clones were identified in each. These were analyzed by restriction mapping and, in several cases, by partial sequence determination. The largest representative clone, which was 3.5 kb in length, was selected for further analysis including complete nucleic acid sequencing on both strands. The cDNA contains a single large open reading frame encoding a 928-amino acid protein with a molecular size of 103.9 kDa (Fig. 1A). The 5`-untranslated sequence contains a single termination codon in-frame with the predicted translation start site, which conforms reasonably well to the Kozak consensus sequence(27) . The 3`-untranslated region includes a poly(A) tail and a sequence from A to A that is similar to the polyadenylation site consensus(28) . 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. However, 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 (see below).



Figure 1: Nucleic acid sequence and deduced amino acid sequence of hDGK. Domain analysis and comparison to rdgA. A, cDNA nucleic acid sequence and the deduced amino acid sequence of hDGK. The zinc fingers are underlined, and key cysteine and histidine residues are marked with a . Serine residues within the MARCKS homology region are marked by an *. The residues within the ATP-binding motif are double underlined. The ankyrin motifs are displayed within boxes. B, a comparison of the predicted protein sequences of hDGK and rdgA. C, the alignment of hDGK carboxyl-terminal sequences with the consensus repeat found in ankyrin (47) . Conservative substitutions were included in the alignment as shown.



The domain structure of hDGK differs significantly from the presently known DAG kinases, particularly the mammalian isozymes alpha, beta, and . The most intriguing difference is the presence of a sequence, KKKKRASFKRKSSKK (Fig. 1A), which is similar to the phosphorylation site of the myristoylated, alanine-rich C-kinase substrate (MARCKS)(29) . MARCKS is a major substrate for protein kinase C, which is activated by DAG. In addition, among the known mammalian isoforms, hDGK uniquely lacks an E-F hand motif, a domain implicated in calcium binding. Moreover, hDGK contains four tandem ankyrin repeats at the carboxyl terminus (Fig. 1C). In contrast to hDGK, ankyrin consistently contains an aspartic acid or an asparagine at position 29 within this motif, whereas hDGK has a methionine at this position in three of the four tandem repeats. Finally, in hDGK, the N-terminal sequence Leu to Glu and internal sequence Pro to Pro scored favorably (9.5 and 16.1, respectively) in a PEST FIND program(30) . PEST sequences are frequently observed in rapidly degraded proteins, suggesting that hDGK may be regulated by protein degradation. Like the other DAG kinases, hDGK contains two zinc finger-like structures in a cysteine-rich region at the amino terminus. The first zinc finger includes the sequence HXCX(6)CXCX(2)CX(4)HX(2)CXC and the second HXCX(2)CXCX(2)CX(4)HX(4)CX(9)C. The hDGK contains a single motif within the catalytic domain that conforms to an ATP binding site (Gly to Lys)(31) .

Expression of hDGK mRNA by Mammalian Cells

We analyzed a variety of mammalian tissues by Northern blot to determine whether, like several other DAG kinases, hDGK has a restricted pattern of expression. Endothelial cells expressed a transcript of 3.7 kb that hybridized to the hDGK riboprobe (Fig. 2A). Furthermore, we screened a human multiple tissue Northern blot and observed that the hDGK transcript was expressed at the highest levels in brain, but with substantial levels in skeletal muscle, heart, and pancreas (Fig. 2B). All other tissues tested showed only low levels of expression. We detected additional transcripts corresponding to 4.2 kb and 9.0 kb in skeletal muscle and heart. These may represent alternatively spliced forms of hDGK or other homologous transcripts. The similarity between hDGK and the rdgA locus suggested that, as with rdgA, the hDGK might have a critical role in retinal function. As a first test of this hypothesis, we probed a Northern blot containing total RNA from human retina, and we were unable to detect significant expression of hDGK (data not shown). Importantly, the positive control of glyceraldehyde-3-phosphate dehydrogenase mRNA from retina confirmed the integrity of the RNA, and, in the same blot, expression of hDGK was detected in endothelial cells. Although subsequent studies with more sensitive methods may reveal expression in the retina, it is clear that the pattern of expression of these homologous genes is very different.


Figure 2: Diacylglycerol kinase is expressed in multiple tissues, but most strongly in brain. The 761-bp PCR product corresponding to hDGK (see ``Results'') was used as a probe in Northern blots of 10 µg of total RNA extracted from cultures of human endothelial cells (HUVEC) (A) or a human multiple tissue Northern blot (Clontech) (B). In lane C, we measured actin mRNA expression in the corresponding lanes of the human multiple tissue Northern blot as a positive control.



Heterologous Expression and Characterization of hDGK

We next examined whether the isolated cDNA encoded a functional DGK activity. COS-7 cells were transfected for 48 h with either vector alone (pcDNA1/AMP) or vector containing the DGK cDNA in the forward orientation (pcDNA1/AMP:DGK). Following transfection, cell homogenates were assayed for DGK activity in the presence of 1,2-dioleoyl-sn-glycerol (Fig. 3). The cells transfected with the DGK cDNA displayed a 267-fold increase in DGK activity, in comparison to vector-transfected cells, confirming that hDGK encodes a functional diacylglycerol kinase. The formation of phosphatidic acid was linear with respect to the amount of protein assayed, and, for the additional characterization, we used 1 µg of cell lysate per assay. The lack of an E-F hand domain in the protein structure suggested that calcium would not influence the activity, and this was confirmed in two ways: the usual assay conditions include EDTA and EGTA and substantial activity was observed (Fig. 3) and, when the assay mixture was supplemented with an excess of calcium (0.1-2 mM) at the start of the reaction, there was no increase in activity (data not shown).


Figure 3: Heterologous expression of hDGK results in elevated levels of diacylglycerol kinase activity. COS-7 cells were transfected with either hDGK (pcDNA1/AMP:DGK; filled circles) or vector alone (pcDNA1/AMP; open circles). After 48 h, the cells were harvested, and the indicated amounts of cellular homogenate were assayed for DGK activity using 1,2-dioleoyl-sn-glycerol as the substrate. This experiment is representative of results observed with cells from two different transfections.



We next examined the substrate specificity of the expressed hDGK (Table 1). A strong preference for 1,2-diacylglycerol over 1,3-diacylglycerol was observed, and hDGK catalyzed the phosphorylation of a short chain diacylglycerol (diC) in preference to all the long chain diacylglycerols examined. This result likely reflects the fact that the short chain substrate is more soluble than the other substrates we tested. Among the long chain diacylglycerols examined, we observed a slight preference for 1-stearoyl-2-arachidonyl-sn-glycerol. Interestingly, the expressed DGK activity did not distinguish between molecular species that varied only in the relative position of the fatty acids: 1-palmitoyl-2-oleoyl-sn-glycerol versus 1-oleoyl-2-palmitoyl-sn-glycerol. The vector-transfected cells did have detectable DGK activity, but it displayed a distinctly different substrate specificity and did not contribute significantly to the activity measured from hDGK-transfected cells (Fig. 3). For all substrates assayed, the DGK activity of vector-transfected COS-7 cells was highest for 1-stearoyl-2-arachidonyl-sn-glycerol with a specific activity of 9.05 nmol/mg/h.



Platelet DAG kinase is inhibited by the compound R59949 which has been used to dissect the role(s) of DGK in cellular responses(32) . We next asked whether R59949 could block the activity of hDGK in vitro. We observed that the expressed hDGK was not significantly inhibited (94% of control) by 100 µM R59949. In contrast, the DGK activity of human platelets was reduced to 54% of control by 100 µM R59949.

To confirm the predicted size of the hDGK protein, we made a polyclonal antibody to a peptide based on the carboxyl-terminal sequence: (C)LENRQHYQMIQREDQE. This antibody was used to probe a Western blot containing protein from hDGK- or vector-transfected COS-7 cells (Fig. 4). Proteins with apparent masses of 117 kDa and 114 kDa were recognized by the polyclonal antibody in hDGK-transfected cells, but were not present in control cells. Furthermore, the recognition of these proteins was blocked by preincubation of the antibody with the corresponding peptide antigen confirming that the interaction of the antibody with these proteins was specific. In subsequent studies, we have detected the expression of endogenous DGK in a glioblastoma-derived human cell line (A-172) by Western blotting (data not shown). Similarly, A-172 cells expressed two immunoreactive proteins which exhibited apparent molecular weights indistinguishable from those detected from transfected COS-7 cells.


Figure 4: COS-7 cells transfected with the hDGK clone express novel proteins. Lanes 1 and 3 contain 25-µg samples of vector (pcDNA1/AMP) transfected COS-7 cells. Lanes 2 and 4 contain 25-µg samples of hDGK (pcDNA1/AMP:DGK) transfected COS-7 cells. Lanes 1 and 2 were probed with the carboxyl-terminal anti-peptide rabbit antibody. Lanes 3 and 4 were probed with the carboxyl-terminal anti-peptide rabbit antibody after preincubation with the corresponding peptide as described under ``Experimental Procedures.''




DISCUSSION

We report here the molecular cloning and characterization of a new human diacylglycerol kinase. Similar to the previously identified DGK isozymes, hDGK contains zinc finger-like sequences and an ATP binding site consensus. In contrast to DGKalpha, -beta, and -, the hDGK isoform lacks an apparent E-F hand motif (33) and its activity is not affected by calcium. This DGK is unique in that it contains a sequence which is homologous to the MARCKS phosphorylation site domain, which may have an important regulatory role(s). The predicted protein sequence is 42% identical and 61% similar to another DGK from Drosophila (dDGK2), which is encoded by the rdgA locus(13) . dDGK2 is the only other DGK that is known to have ankyrin repeats, which are motifs implicated in a variety of functions including protein-protein interactions. There are several significant differences between hDGK and dDGK2, however, including the predicted length of the amino termini, the markedly different patterns of tissue expression, and the presence of a sequence homologous to the MARCKS phosphorylation site domain in hDGK. Northern blot analysis showed that hDGK is expressed as a 3.7-kb message in human endothelial cells, and in human tissues is expressed at highest levels in brain. COS-7 cells transfected with hDGK have elevated levels of DGK activity that are not affected by R59949(32) , a compound that inhibits DGK activity of human platelets. Further analysis of the expressed hDGK activity demonstrated that it lacks substrate specificity among diacylglycerols with long chain fatty acyl groups, but has a clear preference for 1,2-diacylglycerols compared with 1,3-diacylglycerols.

The protein encoded by hDGK is 928 amino acids in length and has a predicted molecular mass of 103.9 kDa. Following expression in COS-7 cells, we observed two separate immunoreactive bands of 117 kDa and 114 kDa by Western blot analysis, which conform reasonably well to the predicted size. In other experiments, utilizing the anti-hDGK peptide antibody, we also detected the expression of two endogenous proteins from a glioblastoma-derived human cell line (A-172) by Western blot analysis that were indistinguishable in size from the immunoreactive proteins expressed by the transfected COS-7 cells (data not shown). The apparent increase in size on SDS-PAGE may be a result of aberrant migration on the gel as the MARCKS protein consistently migrates at a higher molecular weight than expected on SDS-PAGE(29) , and it is possible that the homologous MARCKS phosphorylation site domain in hDGK decreases its mobility. Alternatively, the presence of two bands on the Western blot may indicate post-translational processing or reflect partial proteolysis of the larger band. We believe that the latter is unlikely since the ratio of the two bands has remained consistent in several experiments.

The N terminus of hDGK contains two zinc finger-like sequences: HXCX(6)CXCX(2)CX(4)HX(2)CXC and HXCX(2)CXCX(2)CX(4)HX(4)CX(9)C. Similar domains are represented in protein kinase C(34) , other known DAG kinases (alpha, beta, and ), Raf-1(35) , Vav(36) , n-chimaerin(37) , beta-chimaerin(38) , and Unc-13(39) . These regions have been shown to confer binding properties for diacylglycerol, phorbol esters, and phosphatidylserine as well as zinc (40, 41) . Thus, we conclude that these sequences in hDGK likely comprise the substrate binding regions.

Interestingly, hDGK also contains a sequence carboxyl-terminal to the cysteine-rich region which is homologous to the phosphorylation site domain of the MARCKS protein(29) . The phosphorylation site domain of MARCKS has been demonstrated to bind calmodulin, promote actin aggregation, and regulate intracellular localization in a phosphorylation-dependent manner(42, 43, 44) . hDGK Ser, Ser, and Ser represent potential phosphorylation sites for PKC based on contextual identity to the phosphorylated serines of the MARCKS protein. In related studies, others have observed that a peptide with this sequence is a substrate for PKC, but that it does not bind to calmodulin. (^2)

The catalytic domain of DGK displays strong homology to all previously identified DGK isotypes. Homology between hDGK and dDGK2 are highest in this region displaying 72% similarity and 52% identity. Internal to this domain lies the sequence GXGXXGXK, which conforms to an ATP consensus binding motif(31) . Evidence that this site is required for DGK enzymatic activity derives from the analysis of a rdgA mutation in Drosophila which leads to retinal degeneration. This mutant contains an amino acid substitution (Gly Asp) within the ATP-binding motif(13) , and eye tissue from rdgA homozygotes cannot synthesize phosphatidic acid, as measured by P incorporation(45, 46) .

Four sequential ankyrin repeats were identified at the carboxyl terminus of hDGK. Ankyrin, an integral component of the cytoskeleton, contains 22 N-terminal tandem repeats conforming to a common consensus(47) . Proteins containing ankyrin repeats are involved in a variety of cell regulatory processes including: gene regulation (48, 49, 50) , cell cycle control(51) , and cellular fate determination(52, 53) . The ankyrin domain has been shown to facilitate structural and regulatory protein-protein interactions. The significance of these repeats has been demonstrated by a Drosophila rdgA mutant, which has a nonsense mutation carboxyl-terminal to the catalytic domain that results in a predicted protein that lacks all four ankyrin repeats (13) . Although which (if any) properties of DGK are conferred by the ankyrin repeats remain to be established, it is intriguing that some DGK activities have been reported to be associated with the cytoskeleton(11, 54, 55) .

The sequence of hDGK reveals that it is most closely related to the Drosophila DGK2, derived from the rdgA gene (Fig. 1B). Mutants at this locus exhibit retinal degeneration(46, 56) , and, although the phototransduction cascade of mammalians and invertebrates appear to differ markedly, light-dependent phosphatidylinositol metabolism has been observed in mammalian retina (57) . This suggested that the roles of the two enzymes might be similar. Consistent with this observation, hDGK has been demonstrated to be highly expressed in human retina(10) . However, in contrast to the selective expression of dDGK2 in Drosophila eye, hDGK is expressed in a variety of tissues, and there is no significant expression in human retina. This suggests that dDGK2 and hDGK have different functions. The identification and molecular cloning of this new human isoform of diacylglycerol kinase, hDGK, will help dissect the role of such kinases in signal transduction and lipid metabolism.


FOOTNOTES

*
This work was supported by Grants CA59548 from the National Cancer Institute and HL50153 from the Special Center of Research in ARDS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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) U51477[GenBank].

§
To whom correspondence should be addressed. Tel.: 801-585-3401; Fax: 801-585-6345.

(^1)
The abbreviations used are: DAG, diacylglycerol; DGK, diacylglycerol kinase; diC, 1,2-didecanoyl-sn-glycerol; HUVEC, human umbilical vein endothelial cells; MARCKS, myristoylated alanine-rich C kinase substrate; PKC, protein kinase C; rdgA, retinal degeneration A; TNF, tumor necrosis factor; bp, base pair(s); kb, kilobase(s); PCR, polymerase chain reaction; MOPS, 4-morpholinopropanesulfonic acid; PA, phosphatidic acid.

(^2)
P. J. Blackshear and E. Kennington, personal communication.


ACKNOWLEDGEMENTS

We thank Scott Rogers (University of Utah) for his assistance with the PEST-FIND program. We are grateful to Ralph Whatley, Matt Topham, Li Ding, Jeremy White, and Elie Traer for helpful discussions. Bob Shackman (University of Utah Peptide/DNA User facility, supported by Grant CA42014) and Margaret Robertson (University of Utah DNA Sequencing Core Facility) contributed key technical assistance. We thank Nicolas G. Bazan (LSU Eye Center, New Orleans, LA) for providing a sample of human retina.

Note Added in Proof-During the publication of this manuscript, the identification of another diacylglycerol kinase () was reported. The nomenclature of this manuscript was chosen to include this new member of the diacylglycerol kinase family.


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