INTRODUCTION

Phospholipase D (PLD)¹ catalyzes the hydrolysis of phosphatidylcholine (PC) to phosphatidic acid and choline (1). A variety of signal molecules such as hormones, neurotransmitters, and growth factors are known to induce the activation of PLD in a wide range of cell types. Hence PLD is implicated in a broad spectrum of physiological processes and diseases, including metabolic regulation, inflammation, secretion, mitogenesis, oncogenesis, neural and cardiac stimulation, diabetes, and senescence (for reviews, see Ref. 2). Despite its crucial importance in signal transduction, the molecular structure and characteristics of PLD enzyme are only poorly understood.

Multiple PLD isoforms exist in mammalian tissues. Several factors were reported to stimulate PLD activity in vitro, including unsaturated fatty acid (3), phosphatidylinositol 4,5-bisphosphate (PIP₂) (4), monomeric GTP-binding proteins (G proteins) such as ADP-ribosylation factor 1 (ARF1) (5, 6) and RhoA (7, 8), protein kinase C (9), and calmodulin (10). Massenburg et al. (11) showed that two major forms of PLD activity in rat brain membranes can be separated into ARF-dependent and oleate-dependent enzymes, clearly indicating that these are distinct isoforms. Both oleate-dependent and ARF-dependent types of PLD were recently highly purified from pig lung and brain, respectively (12, 13). In addition, there may be multiple forms of small G protein-dependent PLD including ARF-sensitive, RhoA-sensitive, and ARF-, RhoA-sensitive PLDs. Siddiqi et al. (14) reported that the cytosolic fraction of HL-60 cells contained a soluble PLD activated by ARF, but not RhoA. Malcolm et al. (8) showed rat liver plasma membrane PLD to be sensitive to RhoA, but not to ARF. PLDs in HL-60 membranes and brain membranes are thought to contain the ARF-, RhoA-sensitive type since ARF and RhoA acted on the HL-60 enzyme in a synergistic manner (14-16). PIP₂, another important activator of PLD, was shown to be generally required for the small G protein-dependent PLDs (5, 6, 8, 17), but not required for the oleate-dependent PLD (12). However, Liscovitch et al. (4) demonstrated that the activation of PLD of rat brain membranes will occur without the addition of GTPS. Hence there may be a PIP₂-sensitive, small G protein-insensitive PLD in the brain. To further confirm and characterize these putative PLD isoforms, it is extremely important to purify the enzymes and clone the encoding cDNA.

The cloning of cDNA for higher eukaryote PLD was initially reported by Wang et al. (18), who isolated the cDNA for castor bean (Ricinus communis) PLD by using oligonucleotide probes based on the amino-terminal amino acid sequence of the purified protein (19). Using sequence similarity to castor bean PLD, the Saccharomyces cerevisiae SPO14 gene (20) was identified as the yeast PLD gene (21, 22). By searching for human expressed sequence tags bearing an amino acid sequence similar to the SPO14 sequence, Hammond et al. (23) obtained human cDNA for PLD and designated it hPLD1. Characterization of recombinant hPLD1 revealed that it was activated by PIP₂ and ARF1. The activity was strongly inhibited by oleate in agreement with the data obtained for rat brain ARF-dependent PLD (11). In the present study, we cloned cDNA for a novel type of PLD from rat brain (rPLD2) using highly degenerate PCR primers corresponding to the conserved regions among various eukaryote PLDs. The obtained cDNA showed sequence similarity to hPLD1 and contained a significant deletion in the middle of the sequence. Characterization of the rPLD2 expressed in the fission yeast Schizosaccharomyces pombe revealed that the PLD was stimulated by PIP₂, inhibited by oleate, and thus partially resembled hPLD1. In sharp contrast to hPLD1, however, rPLD2 was not affected by ARF1 or RhoA, indicating that rPLD2 is a novel type of PLD.

EXPERIMENTAL PROCEDURES

Total RNA was extracted from 1 g of male rat brain using TRIZOL reagent (Life Technologies, Inc.) according to the manufacturer's instructions. Poly(A)⁺ RNA was obtained from total RNA using Oligotex-dT30 Super (Japan Roche, Tokyo). Oligo(dT)-primed cDNA synthesis was carried out using a cDNA synthesis kit (Life Technologies, Inc.). cDNAs were ligated to the ZAP II arms (Stratagene, La Jolla, CA), and packaged using GigaPack II Gold packaging extracts (Stratagene).

To obtain the probe for cDNA screening, PCR was performed using a rat brain ExCell cDNA library as the template. Recombinant phages were precipitated with 20% (w/v) polyethylene glycol 6000 and 2 M NaCl, resuspended in 10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA, and treated at 95 °C for 5 min. The first round PCR was performed at 93 °C for 1 min, 55 °C for 2 min, and then 72 °C for 1 min for 35 cycles using the phage solution as the template. The second round PCR was performed under similar conditions using 1:50 volume of the first PCR amplification mixture as the template. The 5-amplimer DBP1 (5-GCIMGICAYTTYRTICARMGITGG-3, where I is inosine, M is A or C, R is A or G, and Y is C or T) corresponded to the amino acid sequence ARHF(V/I)QRW (hPLD1, amino acid residues 694-701 (23); S. cerevisiae PLD1, 888-895 (21); S. pombe open reading frame SPAC2F7.16c, 769-776; Fig. 1). The 3-amplimer DBP2 (5-TCRTTDATRTTIGCISWICCDAT-3, where D is A, G, or T, S is C or G, and W is A or T) corresponded to the amino acid sequence IGSANIN(D/E) (hPLD1, 909-1,016; S. cerevisiae PLD1, 1,109-1,116; S. pombe open reading frame SPAC2F7.16c, 993-1,000; Fig. 1). The amplified 660-base pair fragment (DBT1 in Fig. 1) was gel-purified and cloned into the pGEM-T vector (Promega, Madison, WI).

Plaques were formed using a rat brain ExCell cDNA library, then transferred to Hybond N+ membranes (Amersham Corp.) and screened with digoxigenin-labeled DBT1 as the probe by hybridizing at 68 °C overnight in a solution containing 5 × SSC (1 × SSC = 0.15 M NaCl in 0.015 M sodium citrate), 1% blocking reagent (Boehringer Mannheim), 0.02% SDS, and 0.1% sodium N-lauroylsarcosine. The filters were washed twice with 2 × SSC in 0.1% SDS at room temperature and twice in 0.1 × SSC in 0.1% SDS at 60 °C. Positive phages were located using a digoxigenin-labeled nucleic acid detection kit (Boehringer Mannheim) according to the manufacturer's instructions. Plasmids were obtained from the isolated phages by in vivo excision. The clone DB1 obtained (Fig. 2) was cleaved with PstI (one site within the insert; another site in the vector), and the excised 300-base pair fragment was used as the probe for the second-round screening of a rat brain ZAPII cDNA library to obtain the full-length cDNA (DB3) using similar screening conditions.

Both strands of DNA were sequenced using a DNA sequencing kit (Perkin-Elmer) on the 373A DNA sequencer (Perkin-Elmer) after subcloning into pBluescript II (Stratagene).

Total RNA (0.1 µg) was reverse-transcribed and subjected to PCR amplification using a one-step RT-PCR kit (Toyobo, Osaka) according to the manufacturer's instructions. The reverse transcription was carried out at 60 °C for 30 min, and PCR amplification was performed at 94 °C for 1 min, 56 °C for 1 min, and then at 60 °C for 1.5 min for 40 cycles using primers 5-TCAAGGCCAGATACAAGATACC-3 (rPLD2 sense primer corresponding to positions 2,042-2,063, DBP3 in Fig. 3), 5-CACGTAGACTCGGAAACACTGC-3 (rPLD2 antisense primer corresponding to positions 2,352-2,373, DBP4 in Fig. 3), or 5-TCCACCACCCTGTTGCTGTA-3 (glycerol-3-phosphate dehydrogenase sense primer) and 5-ACCACAGTCCATGCCATCAC-3 (glycerol-3-phosphate dehydrogenase antisense primer).

S. pombe strain TKP1 (h ade6-704 leu1-32 ura4-D18) was used as the host for transformation. Edinburgh minimal medium and YES medium were used for culture (24). Adenine, L-leucine, uracil, and thiamine were added at concentrations of 100, 100, 50, and 5 mg/liter, respectively, where required. Yeast cells were grown aerobically at 32 °C and then transformed by the lithium acetate method as described (25). Standard procedures for S. pombe manipulation were as described (24).

A 3.4-kilobase pair SalI-XbaI fragment (the SalI site was in the vector, and the XbaI site was blunt-ended) containing the entire open reading frame for rPLD2 was isolated from DB3 and subcloned into the SalI/SmaI site of the pREP3X vector to yield pREP3X-rPLD2. pREP3X was a derivative of the S. pombe expression vector pREP1 (26) containing the S. cerevisiae LEU2 marker and a thiamine-repressible promoter.

Yeast cells grown in Edinburgh minimal medium in the presence of thiamine were washed three times with the medium without thiamine, cultured in 100 ml of the same thiamine-free medium for 24 h at 32 °C, and finally harvested by centrifugation at 2,000 × g for 5 min. The cells were washed with water and suspended in 1 ml of the extraction buffer containing 50 mM Hepes/NaOH, pH 7.0, 1 mM EDTA, 1 mM EGTA, 0.1 mM dithiothreitol, 2 µM p-amidinophenylmethanesulfonyl fluoride, and 300 mM sucrose. The cells were disrupted by vigorously vortexing with 1.5 g of glass beads (0.3 mm diameter) four times for 1 min at 4 °C. The beads and the cell debris were removed by centrifugation at 2,000 × g for 10 min. The cell extract thus obtained was further centrifuged at 100,000 × g for 60 min to obtain the cytosolic and membrane fractions. The membrane fraction was suspended in 0.2 ml of the extraction buffer. The samples were kept at 4 °C until use.

The PLD activity in the cell extracts was determined essentially as described (5) by measuring the transphosphatidylation activity in the presence of ethanol. Unless otherwise stated, the cell extracts were incubated with 50 mM Hepes/NaOH, pH 7.5, 200 mM KCl, 200 mM NaCl, 3 mM MgCl₂, 2 mM CaCl₂, 3 mM EGTA, 1 mM dithiothreitol, 400 mM ethanol, 140 µM phosphatidylethanolamine (PE), 12 µM PIP₂, and 8.6 µM 1-palmitoyl-2-[1-¹⁴C]palmitoyl-PC (130 dpm/pmol) (Amersham Corp.) in a total volume of 100 µl for 60 min at 37 °C. PC, PIP₂, and PE were added as the mixed micelles (5). GTPS, ARF1, and RhoA were included as indicated. The reaction was stopped with 100 µl of 1 M HCl. The lipid product was extracted with 1 ml of chloroform/methanol (2:1, by volume) after adding 0.5 ml of 170 mM NaCl. Lipids were separated on a silica gel 60 thin-layer plate (Merck) with chloroform/methanol/acetic acid (13:3:1, by volume). All experiments were performed at least twice, and the representative data were presented.

Recombinant N-myristoylated ARF1 was prepared from Escherichia coli expressing human ARF1 and yeast myristoyl-CoA:protein N-myristoyltransferase (27) as described by Randazzo et al. (28). Recombinant isoprenylated RhoA was prepared from Sf9 cells expressing human RhoA as described by Mizuno et al. (29).

RESULTS

To isolate a novel PLD cDNA from rat brain, we synthesized degenerate primers (DBP1 and DBP2) corresponding to the conserved domain of various eukaryote PLDs (Fig. 1). An open reading frame of S. pombe (SPAC2F7.16c on chromosome I; GenBank^TM Z50142[GenBank]) was also included in the comparison because it closely resembled other eukaryote PLDs and was hence thought to be putative S. pombe PLD. We used the degenerate primers for PCR with a rat brain ExCell cDNA library as the template. The 660-base pair PCR product (DBT1 in Fig. 2) was cloned into the pGEM-T vector, and the obtained clones were subjected to restriction analysis. All of the 48 clones analyzed showed the same restriction pattern, indicating that all clones were identical. Sequence analysis revealed that the sequence significantly resembled that of hPLD1 (23). Thus DBT1 was used to screen a rat brain ExCell cDNA library as the probe. From the 2 × 10⁵ plaques screened, one positive clone was obtained and designated DB1 (3.1 kilobase pairs, Fig. 2). The 5-portion of DB1 was subsequently used as the probe in the second round of screening to obtain the full-length cDNA. Five clones were obtained from 2 × 10⁵ plaques of a rat brain ZAPII cDNA library and analyzed by restriction analysis and in some cases by partial sequencing analysis. Clone DB3, which contained the largest insert (4.6 kilobase pairs, Fig. 2), was selected, and the complete nucleotide sequence was determined on both strands. DB3 contained a single large open reading frame capable of encoding 933 amino acids with a calculated molecular weight of 105,992 (Fig. 3). The 5-untranslated region contained an in-frame termination codon followed by the predicted translation start site conforming to the Kozak consensus (30). The 3-untranslated region contained a poly(A)⁺ tail preceded by the polyadenylation signal at nucleotides 4,530 to 4,539 (31). The predicted amino acid sequence was compared with that of hPLD1 by dot matrix analysis (Fig. 4A). Overall, the sequences of the two proteins are 50% identical, indicating that the protein is structurally related to hPLD1. Notably, there was a significant deletion of about 120 amino acid residues in the middle of the predicted sequence compared with hPLD1. We designated this protein rat phospholipase D2 (rPLD2) since we had obtained a rat cDNA clone encoding a protein 91% identical to hPLD1.² rPLD2 also contained the consensus regions conserved among the eukaryote PLDs (Figs. 1 and 4B). Thus rPLD2 belongs to the same family of protein as hPLD1, yeast PLDs, and castor bean PLD. Of particular interest, the sequence IGSANIN (which was used for designing the PCR primer (Fig. 1)) was perfectly conserved in all of the five eukaryote PLD sequences compared, suggesting the importance of this sequence in the activity and/or structure of eukaryote PLD.

We next sought to express the isolated cDNA using the fission yeast S. pombe as the expression system. It has been shown that many mammalian cDNAs (for example, protein kinase C (32) and phosphatidylinositol 3-kinase (33)) can be efficiently expressed in S. pombe. The major advantages of this system are that transfectants are easy to handle and are stable. Thus S. pombe cells were transfected with either vector alone or rPLD2 cDNA. Cells were disrupted by vortexing with glass beads, fractionated into the cytosolic and membrane fractions, and then assayed for PLD activity using labeled PC embedded in the PIP₂-PE mixed micelles as described by Brown et al. (5). As the most reliable assay of PLD, we measured the transphosphatidylation activity of the enzyme (34-36). The enzyme was incubated with the PC-PIP₂-PE micelles in the presence of ethanol, and the phosphatidylethanol formed was separated by thin-layer chromatography and counted. Although no obviously increased protein band was observed on SDS-polyacrylamide gel electrophoresis, the membrane fraction of the rPLD2 transfectants displayed a 26-fold increase in PLD activity when compared with the vector transfectants, confirming that the cDNA indeed encoded a functional PLD (Fig. 5). The cytosolic fraction from the rPLD2-transfected cells also displayed a 4.4-fold increase in PLD activity. Thus the major portion of the expressed PLD activity was localized in the membrane fraction, although rPLD2 had no extended hydrophobic amino acid stretch as examined by the method of Kyte and Doolittle (37). When the extract from rPLD2 transfectants was incubated in the absence of ethanol, phosphatidic acid was formed instead of phosphatidylethanol (data not shown).

We next examined the enzymatic properties of the rPLD2 enzyme using the membrane fraction of the transfectant. A test mixture was prepared by removing PE and PIP₂ from the standard mixture to examine their effects. As shown in Fig. 6A, PE or PIP₂ were stimulatory to the enzyme. Remarkably, PE and PIP₂ synergistically activated the enzyme. This synergistic effect was only obtained with this combination. When phosphatidylserine was used instead of PE, stimulation by PIP₂ was completely abolished, and phosphatidylinositol was inhibitory. When increasing concentrations of PIP₂ were used in the presence of PE, stimulation occurred at low concentrations of PIP₂ and leveled off at 10 µM (Fig. 6B). The extent of stimulation (5.8-fold) and the required PIP₂ concentration were comparable with those reported previously for PLD activities in mammalian tissues (11, 13, 17) and Sf9-expressed hPLD1 (23).

Many reports implicated ARF1 (5, 6) and RhoA (7, 8) as the regulators of PLD activity. hPLD1 was reported to be activated by ARF1 (23). As shown in Fig. 7, A and B, however, we found that rPLD2 was not activated by either ARF1 or RhoA in the presence of GTPS. This was in sharp contrast to the case of hPLD1, which was sensitive to both ARF1 and RhoA (23). In addition, rat brain cytosol, which is known to stimulate small G protein-dependent PLD very effectively (15, 38, 39), had no stimulatory effect on rPLD2 activity, although it was very effective on the rat brain membrane PLD that was used as the control (Fig. 7C). Thus the response of rPLD2 to the small G proteins was completely different from that of hPLD1. As shown in Fig. 7D, 10⁴ M concentrations of oleic acid strongly inhibited the enzyme as in the case of hPLD1 (23).

GTPS

We examined the expression of rPLD2 mRNA in various rat tissues by RT-PCR with a pair of rPLD2-specific primers (DBP3 and DBP4 in Fig. 3). As shown in Fig. 8, rPLD2 mRNA was expressed in brain, lung, heart, kidney, stomach, small intestine, colon, and testis with the highest level occurring in the lung. Thymus, liver, and muscle expressed relatively low levels of rPLD2 mRNA. The transcript was almost negligible in spleen and pancreas. Although faint, the presence of an additional band seen in liver, kidney, and muscle suggested the occurrence of an alternatively spliced form of rPLD mRNA in these tissues.

DISCUSSION

The present cloning of rPLD2 (a second form of PLD) will give us an important tool for elucidating the function and role of the PC signaling in mammalian cells. The presence of multiple PLD isoforms has been predicted on the basis of the multiplicity of enzyme activators (2) and the presence of multiple forms of short DNA fragments resembling yeast and plant PLD in the DNA data base (23, 40). Recently, Yoshimura et al. (41) isolated three DNA fragments (rat PLDa, rat PLDb, and rat PLDc) that ostensibly represent a partial sequence of different PLD cDNAs by RT-PCR on rat C6 glioma cell RNA using degenerate primers for the conserved regions in hPLD1 and S. cerevisiae PLD1. The sequence of rat PLDc was 99% identical to the rPLD2 sequence reported here, indicating that rat PLDc is a partial sequence of rPLD2. Besides rPLD2, we have isolated two additional full-sized PLD cDNAs from rat brain designated rat PLD1a and rat PLD1b.² Sequence comparison has revealed that rat PLD1a and rat PLD1b probably correspond to rat PLDa and rat PLDb, respectively. Although not yet cloned, rat tissues contain another type of PLD (oleate-dependent enzyme (1, 12)). Thus rat tissues express at least four different PLDs. The existence of such multiple forms of PLD explains why related yet different data have been reported for PLD activities in different cells and tissues. It is of great interest to know the specific roles of individual PLD isoforms in mammalian tissues.

The programs of systematic genome sequencing have supplied numerous new cDNA sequences named expressed sequence tags (42) as well as useful information to search for homologues of the sequence of interest. By searching the human expressed sequence tag library using the BLAST program (43), five GenBank^TM clones (D20091[GenBank], R02092[GenBank], R69739[GenBank], R83570[GenBank], and R93485[GenBank]) were found to have a strong sequence similarity to rPLD2. For example, R93485[GenBank] showed an 84% homology to rPLD2, but only a 63% homology to hPLD1 at the nucleotide level. This clone is thought to be a partial sequence of the human version of rPLD2. Recently, Ribbes et al. (40) searched the expressed sequence tag library for hPLD1 homologues and pointed out that R93485[GenBank] may be a new PLD isoform. Further analysis of R93485[GenBank] will clarify the presence of an rPLD2-type enzyme in human tissues.

Comparison of rPLD2, hPLD1, and other eukaryote PLDs exposed many interesting aspects. Sequence comparison revealed that four distinct regions are conserved in all of the five known eukaryote PLD sequences (rPLD2, hPLD1, S. cerevisiae PLD1, S. pombe putative PLD1, and castor bean PLD (Fig. 4B)). These conserved regions probably play specific roles in the activity and/or structure of PLD. The fourth carboxyl-terminal consensus contains the sequence IGSANIN, which is followed by a highly conserved hydrophilic amino acid stretch (Fig. 1). This sequence is perfectly conserved in all of the compared eukaryote PLDs and also in the rice and maize PLDs recently cloned (44). We are interested in elucidating the functional role of this consensus in the PLD enzymes.

rPLD2 and hPLD1 resemble each other considerably and share a sensitivity to PIP₂ and oleic acid. However, their responses to small G proteins are clearly distinguishable: hPLD1 is stimulated by ARF1 and RhoA, but rPLD2 is not. The major difference in their sequences is a large deletion in the middle of the rPLD2 sequence (Fig. 4A). Hence it is tempting to speculate that this part of the hPLD1 sequence might be responsible for binding to and/or activation by ARF1 and RhoA. Sequence comparison is also expected to provide useful information as to the PIP₂ binding site in the PLD enzyme. Like hPLD1 and rPLD2, S. cerevisiae PLD is sensitive to PIP₂ (21). Thus rPLD2, hPLD1, and S. cerevisiae PLD1 are thought to commonly contain the PIP₂-binding site. There are six conserved regions shared by these three sequences, at least one of which should contain the PIP₂-binding site. This information should be useful for future identification of the PIP₂-binding region in rPLD2 as well as in hPLD1. Interestingly, these regions are also present in the S. pombe putative PLD1 sequence, although PIP₂ stimulation has not yet been reported for S. pombe PLD.