Mammalian Mg2+-independent Phosphatidate Phosphatase (PAP2) Displays Diacylglycerol Pyrophosphate Phosphatase Activity*

(Received for publication, December 19, 1996, and in revised form, February 14, 1997)

Deirdre A. Dillon Dagger , Xiaoming Chen Dagger , Geri Marie Zeimetz Dagger , Wen-I. Wu Dagger , David W. Waggoner §, Jay Dewald §, David N. Brindley § and George M. Carman Dagger

From the Dagger  Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University, New Brunswick, New Jersey 08903 and the § Signal Transduction Laboratories, Lipid and Lipoprotein Research Group, and Department of Biochemistry, University of Alberta, 357 Heritage Medical Research Centre, Edmonton, Alberta, T6G 2S2 Canada

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Recent studies indicate that the metabolism of diacylglycerol pyrophosphate (DGPP) is involved in a novel lipid signaling pathway. DGPP phosphatases (DGPP phosphohydrolase) from Saccharomyces cerevisiae and Escherichia coli catalyze the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyze the dephosphorylation of PA to yield diacylglycerol. We demonstrated that the Mg2+-independent form of PA phosphatase (PA phosphohydrolase, PAP2) purified from rat liver catalyzed the dephosphorylation of DGPP. This reaction was Mg2+-independent, insensitive to inhibition by N-ethylmaleimide and bromoenol lactone, and inhibited by Mn2+ ions. PAP2 exhibited a high affinity for DGPP (Km = 0.04 mol %). The specificity constant (Vmax/Km) for DGPP was 1.3-fold higher than that of PA. DGPP inhibited the ability of PAP2 to dephosphorylate PA, and PA inhibited the dephosphorylation of DGPP. Like rat liver PAP2, the Mg2+-independent PA phosphatase activity of DGPP phosphatase purified from S. cerevisiae was inhibited by lyso-PA, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse PAP2 showed homology to DGPP phosphatases from S. cerevisiae and E. coli, especially in localized regions that constitute a novel phosphatase sequence motif. Collectively, our work indicated that rat liver PAP2 is a member of a phosphatase family that includes DGPP phosphatases from S. cerevisiae and E. coli. We propose a model in which the phosphatase activities of rat liver PAP2 and the DGPP phosphatase of S. cerevisiae regulate the cellular levels of DGPP, PA, and diacylglycerol.


INTRODUCTION

PA1 phosphatase (3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) catalyzes the dephosphorylation of PA to yield DG and Pi (1). Two forms of PA phosphatase exist in mammalian cells. Data indicate that one form of PA phosphatase (PAP1) is primarily responsible for the synthesis of phospholipids and triacylglycerols (2-5), whereas the other form of PA phosphatase (PAP2) is primarily involved in lipid signaling pathways (4-7). PA, the substrate of the PA phosphatase reaction, regulates the activity of several lipid-dependent enzymes (8-12) and exhibits mitogenic effects in mammalian cells (12-16). Thus, the action of PA phosphatase is thought to attenuate the signaling functions of PA (7). In addition, Brindley and co-workers (7, 17) have shown that PAP2, purified from rat liver, has the ability to dephosphorylate LPA, SPP, and CerP. These substrates and their hydrolysis products have been shown to play a role in signaling pathways in mammalian cells (7, 17).

The two forms of PA phosphatase have distinguishing enzymological properties that are used to differentiate them. PAP1 has a Mg2+ ion requirement and is inhibited by the thioreactive agent NEM (4, 6, 7). PAP2 does not have a Mg2+ ion requirement and is insensitive to NEM (4, 6, 7). PAP2, purified from rat liver (18-20) and porcine thymus (21, 22), shares enzymological properties that are strikingly similar to a PA phosphatase activity exhibited by a DGPP phosphatase (DGPP phosphohydrolase) that has recently been isolated from Saccharomyces cerevisiae (23) and Escherichia coli (24). DGPP phosphatase catalyzes the dephosphorylation of the novel lipid DGPP to form PA and Pi (23, 25). When DGPP is supplied as a substrate in vitro, the enzyme removes the beta  phosphate of DGPP to generate PA and then removes the phosphate of PA to generate DG (23, 24). Although DGPP phosphatase utilizes PA as a substrate in the absence of DGPP, the enzyme has a preference for DGPP as a substrate (23, 24). This PA phosphatase activity (23) is distinctly different from that of the PAP1 enzymes that have been purified from S. cerevisiae (26, 27) but does resemble that of the mammalian PAP2 enzymes. Like PAP2, the PA phosphatase activity catalyzed by DGPP phosphatase is Mg2+-independent and NEM-insensitive (23, 24). In addition, the PAP2 (18) and DGPP phosphatase (23, 24) enzymes can utilize LPA as a substrate. Given these similarities, we hypothesized that mammalian PAP2 would display DGPP phosphatase activity. Using purified PAP2 from rat liver we demonstrated that PAP2 catalyzed the DGPP phosphatase reaction. Recent data indicate that DGPP and the enzymes responsible for its metabolism are involved in a novel lipid signaling pathway (23, 28, 29). The implications of PAP2 having DGPP phosphatase activity are discussed in relation to lipid signaling pathways.


EXPERIMENTAL PROCEDURES

Materials

All chemicals were reagent grade. Radiochemicals and EN3HANCE were from DuPont NEN. Scintillation counting supplies were from National Diagnostics. Nucleotides, NEM, Triton X-100, and bovine serum albumin were purchased from Sigma. Phospholipids were purchased from Avanti Polar Lipids and Sigma. Protein assay kits were purchased from Bio-Rad (Coomassie Blue) and Pierce (BCA). Silica gel 60 thin-layer chromatography plates were from EM Science. Protein A-Sepharose and Sephacryl S-200 were from Pharmacia Biotech Inc. E. coli DG kinase was obtained from Lipidex Inc. or Calbiochem. Bromoenol lactone [(E)-6-(bromomethylene) tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one)] was obtained from Dr. Edward A. Dennis (University of California, San Diego, CA).

Preparation of Enzymes

PAP2 (anionic form) was purified from rat liver plasma membranes as described by Waggoner et al. (18). DGPP phosphatase (23) and the 104-kDa Mg2+-dependent PA phosphatase (26, 27) were purified from the microsomal fraction of S. cerevisiae as described previously. PA kinase was purified from plasma membranes of Catharanthus roseus cells as described by Wissing and Behrbohm (30).

Immunoprecipitation and Immunoblotting

Nondenaturing immunoprecipitation (18) and immunoblotting procedures (31, 32) were performed with anti-PAP2 antibodies (Ab-D503) generated against the homogeneous cationic PAP2 from rat liver (18).

Preparation of Substrates

DGPP standard and 32P-labeled (alpha -32P and beta -32P) DGPP were synthesized enzymatically using purified C. roseus PA kinase as described by Wu et al. (23). [32P]PA and [32P]CerP were synthesized enzymatically from DG and long-chain ceramide, respectively, using E. coli DG kinase (33) as described previously (17, 26). Unlabeled CerP was also synthesized enzymatically via the DG kinase reaction (17). SPP was prepared from CerP by acid hydrolysis (34).

Preparation of Triton X-100/Lipid-mixed Micelles

Lipids in chloroform were transferred to a test tube, and solvent was removed in vacuo for 40 min. Triton X-100/lipid-mixed micelles were prepared by adding various amounts of a 5% (w/v) solution of Triton X-100 to the dried lipids. After the addition of Triton X-100, the mixture was vortexed. The surface concentration of lipids in mixed micelles was varied by the addition of Triton X-100. The total lipid concentration in Triton X-100/lipid-mixed micelles did not exceed 20 mol % to ensure that the structure of the mixed micelles was similar to the structure of pure Triton X-100 (35, 36). The uniformity of Triton X-100/DGPP-mixed micelles was determined by Sephacryl S-300 gel filtration chromatography (37). The mole percent of a lipid in a mixed micelle was calculated using the formula: mol %lipid = ([lipid (bulk)]/[lipid (bulk)] + [Triton X-100]) × 100.

Enzyme Assays

DGPP phosphatase activity was measured by following the release of water-soluble 32Pi from chloroform-soluble [beta -32P]DGPP (5,000-10,000 cpm/nmol) or by following the formation of [32P]PA from [alpha -32P]DGPP (2,000-5,000 cpm/nmol) as described by Wu et al. (23). The reaction mixture contained 50 mM Tris-maleate buffer (pH 6.5), enzyme protein, and the indicated concentrations of Triton X-100 and DGPP in a total volume of 50 µl. Kinetic experiments were performed with 50 mM citrate buffer (pH 5.5). The chloroform-soluble phospholipid product of the reaction, PA, was analyzed with standard PA and DGPP by thin-layer chromatography on potassium oxalate-treated plates using the solvent system chloroform/acetone/methanol/glacial acetic acid/water (50:15:13:12:4) (23). The positions of the labeled phospholipids on the chromatograms were determined by autoradiography. The amount of labeled phospholipids was determined by scintillation counting.

Mg2+-independent PA phosphatase and Mg2+-dependent PA phosphatase activities were measured by following the release of water-soluble 32Pi from chloroform-soluble [32P]PA (10,000 cpm/nmol) (18, 38). The reaction mixture for Mg2+-independent PA phosphatase contained 50 mM Tris-maleate buffer (pH 6.5), enzyme protein, and the indicated concentrations of Triton X-100 and PA in a total volume of 50 µl. Kinetic experiments were performed with 50 mM citrate buffer (pH 5.5). The reaction mixture for Mg2+-dependent PA phosphatase contained 50 mM Tris-maleate buffer (pH 7.0), 2 mM MgCl2, 0.1 mM PA, 1 mM Triton X-100, 10 mM 2-mercaptoethanol, and enzyme protein in a total volume of 0.1 ml.

CerP phosphatase activity was measured by following the release of water-soluble 32Pi from chloroform-soluble [32P]CerP (10,000 cpm/nmol) (17). The reaction mixture contained 100 mM Tris-maleate buffer (pH 6.5), 1 mM EDTA, enzyme protein, and the indicated concentrations of Triton X-100 and CerP in a total volume of 25 µl.

All enzyme assays were conducted for 15 min at 30 °C in triplicate. The average S.D. of the assays was ± 5%. The enzyme reactions were linear with time and protein concentration. A unit of enzymatic activity was defined as the amount of enzyme that catalyzed the formation of 1 µmol of product/min. Specific activity was defined as units/mg of protein. Protein concentration was determined using the BCA assay or by the method of Bradford (39) using bovine serum albumin as the standard.

Analysis of Kinetic Data

Kinetic data were analyzed according to the Michaelis-Menten and Hill equations using the EZ-FIT Enzyme Kinetic Model Fitting Program (40). EZ-FIT uses the Nelder-Mead Simplex and Marquardt/Nash nonlinear regression algorithms sequentially and tests for the best fit of the data among different kinetic models.


RESULTS

Rat Liver PAP2 Displays DGPP Phosphatase Activity

Rat liver PAP2 was examined for its ability to catalyze the removal of the beta  phosphate from DGPP using [alpha -32P]DGPP as the substrate. The chloroform-soluble product of the reaction was analyzed by thin-layer chromatography followed by autoradiography. The enzyme catalyzed a time-dependent conversion of DGPP to PA (Fig. 1A). Quantification of the PA spots on the thin-layer chromatogram by scintillation counting showed that the DGPP phosphatase reaction was linear (Fig. 1B). A DGPP phosphatase reaction was also conducted over the same time period using [beta -32P]DGPP as the substrate. In this case, enzyme activity was followed by measuring the water-soluble product of the reaction by scintillation counting. The amount of beta -labeled Pi produced in the reaction was also linear with time and paralleled the amount of PA produced in the reaction using the alpha -labeled substrate (data not shown). Because rat liver PAP2 catalyzes the dephosphorylation of PA (18), we examined the water-soluble fraction of the DGPP phosphatase reaction for the production of alpha Pi. Over the time period of the reaction using the alpha -labeled substrate, only a negligible amount of the alpha Pi was produced (Fig. 1B). Thus, over the standard assay time of the reaction, PAP2 catalyzed essentially the stoichiometric conversion of DGPP to PA and Pi. When the DGPP phosphatase reaction was followed for longer time intervals (e.g., 1 h), the enzyme then removed the phosphate of PA to produce DG (data not shown).


Fig. 1. Time dependence of the DGPP phosphatase reaction catalyzed by rat liver PAP2. DGPP phosphatase activity was measured with 0.1 mM [alpha -32P]DGPP and 5 mM Triton X-100 using 40 ng of rat liver PAP2. After incubation for the indicated time intervals, the chloroform-soluble 32P-labeled product PA was separated from [alpha -32P]DGPP by thin-layer chromatography (A) and then analyzed by scintillation counting (B). The water-soluble 32Pi hydrolyzed from [alpha -32P]DGPP was analyzed by scintillation counting (B). The positions of standard DGPP and PA after thin-layer chromatography are indicated in A.
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Although the rat liver PAP2 preparation used in these studies was highly purified, it was not a homogeneous protein sample (18). We obtained a small sample of homogeneous enzyme from this preparation by immunoprecipitation with anti-PAP2 antibodies (18). This immunoprecipitated enzyme catalyzed the DGPP phosphatase reaction. Because the amount of the immunoprecipitated enzyme was limiting, we examined the properties of the DGPP phosphatase reaction using the nonhomogeneous but highly purified PAP2 preparation (18).

Properties of the DGPP Phosphatase Activity of Rat Liver PAP2

The characteristic properties of rat liver PAP2 are the absence of any divalent cation requirement and its insensitivity to inhibition by the thioreactive compound NEM (7, 18). The DGPP phosphatase activity of the rat liver PAP2 was not dependent on any divalent cation under standard assay conditions. Moreover, the addition of 1 mM EDTA plus 1 mM EGTA to the assay system did not affect activity. DGPP phosphatase activity was insensitive to inhibition by NEM at concentrations up to 10 mM. As a control, the PA phosphatase activity of PAP2 was shown not to require a divalent cation and was insensitive to treatment with NEM. The rat liver PAP1 enzyme is totally inhibited by 2 mM NEM (6).

Balsinde and Dennis (5) have recently discovered a new property that can be used to characterize the differences between the mammalian Mg2+-independent and Mg2+-dependent PA phosphatase activities. Using P388D1 macrophages, these workers have shown that the Mg2+-dependent enzyme is potently inhibited by bromoenol lactone (IC50 = 8 µM), whereas the Mg2+-independent enzyme is insensitive to this reagent (5). We examined if the DGPP phosphatase and PA phosphatase activities of rat liver PAP2 were sensitive to bromoenol lactone. Samples of the purified enzyme were preincubated with increasing concentrations of bromoenol lactone for 10 min and then assayed for each activity. Concentrations of bromoenol lactone up to 100 µM had no effect on the DGPP phosphatase and PA phosphatase activities of PAP2. Bromoenol lactone had no effect on S. cerevisiae DGPP phosphatase activity. As a positive control, we examined the effect of bromoenol lactone on the 104-kDa Mg2+-dependent PA phosphatase purified from S. cerevisiae. As described previously (5), this Mg2+-dependent enzyme was inhibited by bromoenol lactone.

A characteristic property of the DGPP phosphatases isolated from S. cerevisiae (23) and from E. coli (24) is the inhibition of their activities by Mn2+ ions. We examined the effect of Mn2+ ions on the DGPP phosphatase and PA phosphatase activities of rat liver PAP2. The addition of Mn2+ ions to the assay system for DGPP phosphatase resulted in a dose-dependent inhibition of activity (Fig. 2A). The inhibition of DGPP phosphatase activity by Mn2+ ions followed positive cooperative kinetics (n = 3.3). An IC50 value of 0.27 mM was calculated based on the analysis of the data according to the Hill equation. On the other hand, the PA phosphatase activity of PAP2 was relatively insensitive to inhibition by Mn2+ ions up to a concentration of 2.5 mM (Fig. 2B).


Fig. 2. Effect of MnCl2 on the DGPP phosphatase and PA phosphatase activities of rat liver PAP2. The DGPP phosphatase (A) and PA phosphatase (B) activities of rat liver PAP2 (40 ng) were measured with 0.1 mM DGPP and 0.1 mM PA, respectively, and 5 mM Triton X-100 in the absence and presence of the indicated concentrations of MnCl2. The inset in A is a replot of the MnCl2-mediated inhibition of DGPP phosphatase activity.
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Jamal et al. (6) have previously shown that the pH optimum for the Mg2+-independent PA phosphatase activity associated with plasma membranes of rat liver cells was 6.5. However, the pH dependence of the purified enzyme activity has not been examined (18). In this study we examined the effect of pH on the DGPP phosphatase and PA phosphatase activities of purified rat liver PAP2. Maximum DGPP phosphatase activity was observed between pH 5.0 and 6.0, and maximum PA phosphatase activity was observed between pH 5.0 and 6.5. The Mg2+-independence and insensitivity to NEM and bromoenol lactone of the DGPP phosphatase and PA phosphatase activities were not affected when measured at pH 5.5.

Kinetics of the DGPP Phosphatase Activity of Rat Liver PAP2

The kinetics of DGPP phosphatase and PA phosphatase activities of rat liver PAP2 were examined using Triton X-100/phospholipid-mixed micelles. The kinetic analyses of these activities using mixed micelle substrates required that these micelles were homogeneous in size. Previous studies have shown that Triton X-100 forms uniform mixed micelles with PA (37). It was important for us to demonstrate that Triton X-100 formed uniform mixed micelles with DGPP. Gel filtration analysis of a mixture of Triton X-100 plus 5 mol % DGPP showed that Triton X-100 formed uniform mixed micelles with DGPP. The Triton X-100/phospholipid-mixed micelle system permitted the kinetic analyses of DGPP phosphatase and PA phosphatase activities using surface dilution kinetics (41). Surface dilution kinetics is a model system that mimics the physiological surface of the membrane where two-dimensional surface interactions occur (41). Accordingly, the concentrations of DGPP and PA in the mixed micelles were expressed as a surface concentration (in mol %) as opposed to a molar concentration (41). In addition, these activities were independent of the molar concentrations of DGPP and PA at the Triton X-100/phospholipid-mixed micelle concentrations used in this study (17, 23). DGPP phosphatase activity displayed by PAP2 exhibited saturation kinetics with respect to the surface concentration of DGPP (Fig. 3A). The Vmax was 1.24 µmol/min/mg, and the Km value for DGPP was 0.04 mol %. As described previously (17), the PA phosphatase activity of PAP2 displayed saturation kinetics with respect to the surface concentration of PA (Fig. 3B). The Vmax was 0.93 µmol/min/mg, and the Km was 0.04 mol %. The specificity constants (Vmax/Km) for DGPP and PA were 31 and 23, respectively.


Fig. 3. Kinetics of the DGPP phosphatase and PA phosphatase activities of rat liver PAP2. The DGPP phosphatase (A) and PA phosphatase (B) activities of rat liver PAP2 (40 ng) were measured as a function of the surface concentration (mol %) of [beta -32P]DGPP and [32P]PA, respectively. The molar concentrations of DGPP (A) and PA (B), respectively, were held constant at 0.1 mM, whereas the Triton X-100 concentration was varied.
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Effects of PA and DGPP on the DGPP Phosphatase and PA Phosphatase Activities, Respectively, of Rat Liver PAP2

Because rat liver PAP2 utilized both DGPP and PA as substrates, we examined whether PA affected DGPP phosphatase activity and whether DGPP affected PA phosphatase activity. In these experiments, we used a surface concentration of 0.05 mol % of DGPP for the DGPP phosphatase reaction and a surface concentration of 0.05 mol % of PA for the PA phosphatase reaction. These surface concentrations were near the respective Km values for these substrates. Thus, we could readily observe inhibitory or stimulatory effects of PA and DGPP on DGPP phosphatase and PA phosphatase activities, respectively. PA inhibited the DGPP phosphatase activity in a dose-dependent manner (Fig. 4A). An IC50 value for PA of 0.07 mol % was calculated from a replot of the log of relative DGPP phosphatase activity versus the PA concentration. DGPP inhibited the PA phosphatase activity in a dose-dependent manner (Fig. 4B). The IC50 value for DGPP was calculated to be 0.05 mol %.


Fig. 4. Effects of PA and DGPP on the DGPP phosphatase and PA phosphatase activities, respectively, of rat liver PAP2. A, the DGPP phosphatase activity of rat liver PAP2 (40 ng) was measured with 0.05 mol % of [beta -32P]DGPP as substrate in the presence of the indicated surface concentrations of PA. B, the PA phosphatase activity of rat liver PAP2 (40 ng) was measured with 0.05 mol % of [32P]PA as substrate in the presence of the indicated surface concentrations of DGPP.
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Effects of LPA, SPP, and CerP on the S. cerevisiae DGPP Phosphatase

The Mg2+-independent PA phosphatase activity of rat liver PAP2 has been shown to be inhibited by LPA, SPP, and CerP (17). These lipid phosphate compounds can also serve as substrates for PAP2 in vitro (17). Given the similarities between rat liver PAP2 and the S. cerevisiae DGPP phosphatase, we examined whether the Mg2+-independent PA phosphatase activity of S. cerevisiae DGPP phosphatase was inhibited by LPA, SPP, and CerP. The surface concentration of PA (2.2 mol %) used in these experiments was the concentration of PA at its Km value for this reaction (23). All three of these compounds inhibited PA phosphatase activity in dose-dependent manners (Fig. 5). LPA was the most potent inhibitor with an IC50 value of 0.4 mol % (Fig. 5). We also examined the effect of LPA on the DGPP phosphatase activity of the S. cerevisiae enzyme. The surface concentration of DGPP (0.5 mol %) used in this experiment was the concentration of DGPP near its Km value (23). LPA inhibited the DGPP phosphatase activity of the enzyme (Fig. 6). However, the inhibitory effect of LPA (IC50 = 3.3 mol %) on the DGPP phosphatase reaction was much less potent when compared with its inhibitory effect with respect to the PA phosphatase reaction (Fig. 5). As described previously (23), PA did not inhibit the DGPP phosphatase activity of the enzyme (Fig. 6).


Fig. 5. Effects of LPA, SPP, and CerP on the Mg2+-independent PA phosphatase activity of S. cerevisiae DGPP phosphatase. The PA phosphatase activity of the S. cerevisiae DGPP phosphatase (0.5 ng) was measured with 2.2 mol % of [32P]PA (0.1 mM) as substrate in the presence of the indicated surface concentrations of LPA, SPP, and CerP. The concentration of Triton X-100 in the reaction mixture without LPA, SPP, and CerP was 4.5 mM.
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Fig. 6. Effects of LPA and PA on S. cerevisiae DGPP phosphatase activity. The DGPP phosphatase activity of the S. cerevisiae DGPP phosphatase (0.5 ng) was measured with 0.5 mol % of DGPP (0.1 mM) in the presence of the indicated surface concentrations of LPA and PA. The concentration of Triton X-100 in the reaction mixture without LPA and PA was 19.5 mM.
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The inhibition of the S. cerevisiae DGPP phosphatase by LPA, SPP, and CerP suggested that these lipid phosphate compounds might serve as substrates for the enzyme. Indeed, we have recently shown that LPA is a substrate for the DGPP phosphatase (24). DGPP phosphatase catalyzed the dephosphorylation of CerP in a dose-dependent manner (Fig. 7), demonstrating that CerP was also a substrate for the enzyme. The reaction followed saturation kinetics, and the Vmax and Km values were calculated to be 110 µmol/min/mg and 4 mol %, respectively.


Fig. 7. Kinetics of the CerP phosphatase activity of S. cerevisiae DGPP phosphatase. The CerP phosphatase activity of S. cerevisiae DGPP phosphatase (0.5 ng) was measured as a function of the surface concentration (mol %) of [32P]CerP. The molar concentration of CerP was held constant at 0.1 mM, whereas the Triton X-100 concentration was varied.
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DISCUSSION

DGPP phosphatase is a recently discovered enzyme that has been identified in C. roseus, E. coli, S. cerevisiae, rat liver, pig liver, pig brain, and bovine brain (25). The discovery of DGPP phosphatase in such a wide range of organisms suggests that it plays an important role in phospholipid metabolism and cell growth. DGPP phosphatase has been purified to homogeneity from S. cerevisiae and characterized with respect to its enzymological and kinetic properties (23). DGPP phosphatase has been partially purified from E. coli and shown to be the product of the pgpB gene (24). The DGPP phosphatases isolated from S. cerevisiae (23) and E. coli (24) catalyze the dephosphorylation of the beta  phosphate of DGPP to yield PA and then catalyze the dephosphorylation of the PA product to yield DG. The DGPP phosphatase and PA phosphatase activities of the DGPP phosphatase enzymes from S. cerevisiae (23) and E. coli (24) are Mg2+-independent and NEM-insensitive (23, 24). In addition, these DGPP phosphatase activities are potently inhibited by Mn2+ ions (23, 24).

In this study we examined the hypothesis that PAP2 purified from rat liver would display DGPP phosphatase activity. One impetus for this study was the fact that the characteristic properties of PAP2 (i.e. Mg2+-independent and NEM-insensitive PA phosphatase activity) (4, 6, 18, 19, 21) are the same as those of the PA phosphatase activities described for the DGPP phosphatase enzymes from S. cerevisiae (23) and E. coli (24). We showed in this study that PAP2 did indeed catalyze the DGPP phosphatase reaction. Like the DGPP phosphatases from S. cerevisiae (23) and E. coli (24), the DGPP phosphatase activity of PAP2 was Mg2+-independent, NEM-insensitive, and inhibited by Mn2+ ions. The latter effect probably reflects a specific interaction of DGPP with Mn2+ ions that is not exhibited with PA. The rat liver and S. cerevisiae DGPP phosphatase activities were also insensitive to inhibition by bromoenol lactone, which has recently been used to distinguish between the Mg2+-dependent and Mg2+-independent forms of PA phosphatase (5). Furthermore, the S. cerevisiae (23, 24) and E. coli (24, 42, 43) DGPP phosphatases and the rat liver PAP2 (17) were similar in that they used other lipid phosphate compounds such as LPA and CerP as substrates.

Although our studies revealed similarities between rat liver PAP2 and the DGPP phosphatases of S. cerevisiae and E. coli, there were also differences among these enzymes. Interestingly, the affinity of PAP2 for DGPP and PA as substrates was much greater than the affinities of the DGPP phosphatases of S. cerevisiae and E. coli for these substrates (Table I). The specificity constant (Vmax/Km) of rat liver PAP2 for DGPP was slightly higher (1.3-fold) than that for PA (Table I). PA inhibited the dephosphorylation of DGPP by rat liver PAP2, and DGPP inhibited the dephosphorylation of PA by PAP2. The inhibitor constants for PA and DGPP of rat liver PAP2 were similar, and these constants were similar to their respective Km values as substrates (Table I). Thus, the specificity of PAP2 for DGPP and PA was essentially the same. On the other hand, the DGPP phosphatases from S. cerevisiae (23) and E. coli (24) clearly demonstrate a preference for DGPP as a substrate. For these enzymes, the specificity constants for DGPP are about 9-fold higher than those for PA (Table I) (23, 24). The PA phosphatase activity of the S. cerevisiae DGPP phosphatase is potently inhibited by DGPP (Table I) (23). However, concentrations of PA up to 16-fold greater than the concentration of DGPP did not inhibit S. cerevisiae DGPP phosphatase activity.

Table I.

Kinetic constants for rat liver PAP2 and DGPP phosphatases from S. cerevisiae and E. coli


Enzyme DGPP
PA
Vmax Km Vmax/Kma IC50 Vmax Km Vmax/Kma IC50

units/mg mol% mol% units/mg mol% mol%
PAP2 (rat liver) 1.24 0.04 31 0.05b 0.93 0.04 23 0.07c
DGPP phosphatased (S. cerevisiae) 172 0.55 313 0.35b 70 2.2 32 NIe
DGPP phosphatasef (E. coli) 2.16 2.3 0.94 NDg 0.31 3.1 0.1 NDg

a Because the enzymes from rat liver and E. coli have not been purified to homogeneity, the specificity constants reported in the table cannot be compared.
b Inhibitor constant with respect to PA as the substrate.
c Inhibitor constant with respect to DGPP as the substrate.
d Data taken from Ref. 23.
e NI, not inhibitory.
f Data taken from Ref. 24.
g ND, not determined.

A cDNA encoding for PAP2 has been cloned from mouse cells that encodes for a protein with a predicted minimum subunit molecular mass of 31.9 kDa (22). DGPP phosphatase activity is associated with a 34-kDa protein that we have purified to homogeneity from S. cerevisiae (23). We have obtained sufficient amino acid sequence information from this 34-kDa protein to identify and isolate the gene (GenBankTM accession no. U51031[GenBank]) encoding for this enzyme.2 We refer to this gene as DPP1 (diacylglycerol pyrophosphate phosphatase). In addition, we have identified and isolated a second gene (GenBankTM accession no. U33057[GenBank]) from S. cerevisiae that is homologous to DPP1 that we refer to as DPP2.2 The predicted minimum subunit molecular masses of the proteins encoded by these genes are 33.5 and 31.6 kDa, respectively. The subunit molecular mass of the protein encoded by the E. coli pgpB gene is 28 kDa (42). The amino acid sequences of DGPP phosphatases of S. cerevisiae and E. coli and the mouse PAP2 proteins show homology to each other. In particular there are localized regions of high homology that constitute a novel phosphatase sequence motif (44). This motif contains three domains (44). The alignment of the amino acid sequences of PAP2 and the DGPP phosphatases in these domains is shown in Table II. The size of mammalian PAP2 seems to vary in different tissues (18). These different sizes may be attributed to variations in the extent of their glycosylation because treatment of purified PAP2 from rat liver (18) and pig thymus (22) with N-glycanase decreases their apparent size from 51-53 kDa and 35 kDa, respectively, to about 30 kDa. There is no evidence of N-glycosylation of the DGPP phosphatases from S. cerevisiae and E. coli. Collectively, the work presented here indicated that mammalian PAP2 is a member of a phosphatase family that includes DGPP phosphatases from S. cerevisiae and E. coli.

Table II.

Protein alignments of the phosphatase sequence motif of mouse PAP2 and DGPP phosphatases from S. cerevisiae and E. colia 2


Protein        Domain 1b      Domain 2         Domain 3 

PAP2 (mouse) 119-KYTIGSLRP-39-YSGH-44-SRVSDYKHHWSD-283
DGPP phosphatase 1 (S. cerevisiae) 117-KNWIGRLRP-39-PSGH-46-SRTQDYRHHFVD-289
DGPP phosphatase 2 (S. cerevisiae) 135-KLIIGNLRP-41-PSGH-38-SRVIDHRHHWYD-275
DGPP phosphatase (E. coli)  96-KDKVQEPRP-54-PSGH-36-SRLLLGMHWPRD-254

a Data taken from Ref. 44.
b The numbers preceding domain 1 indicate the length in amino acids of the N terminus of the protein. The numbers between domains indicate the amino acids between each domain. The numbers after domain 3 indicate the total amino acids in each protein.

DGPP is a novel phospholipid that was first identified as the product of the PA kinase reaction in the plant C. roseus (28). DGPP has since been found in a variety of plants (29, 30) and in S. cerevisiae (23). The amounts of DGPP in plants and in wild-type S. cerevisiae are barely detectable (23, 29). For example, DGPP accounts for only 0.18 mol % of the major phospholipids in S. cerevisiae (23). The low amount of DGPP is reminiscent of other lipid signaling molecules such as the inositol-containing phospholipids (45-49). Recent studies have shown that DGPP accumulates in plant tissues upon G protein activation through the stimulation of PA kinase activity (29), and metabolic labeling studies have shown that DGPP is metabolized to PA and then to DG (25). At the present time the function of DGPP in phospholipid metabolism and cell signaling is unknown. It has been suggested that DGPP might attenuate the signaling functions of PA, that DGPP is the precursor of the PA that serves as a signaling molecule, or that DGPP itself might function as a signaling molecule (23, 29). PAP2 and the DGPP phosphatases from S. cerevisiae and E. coli are fascinating enzymes in that the product of one reaction becomes the substrate for another reaction (Fig. 8). Based on available information, we propose models in which feedback inhibition of the activities displayed by mammalian PAP2 (Fig. 8A) and S. cerevisiae DGPP phosphatase (Fig. 8B) could regulate the cellular levels of DGPP, PA, and DG. For example, our data showed that for rat liver PAP2, PA potently inhibited the ability of the enzyme to dephosphorylate DGPP and that DGPP potently inhibited the ability of the enzyme to dephosphorylate PA. Although DGPP inhibits the ability of the yeast DGPP phosphatase to dephosphorylate PA (23), its ability to dephosphorylate DGPP is not inhibited by PA.


Fig. 8. Models for the regulation of the reactions catalyzed by rat liver PAP2 and DGPP phosphatase of S. cerevisiae. The figure shows the structures of DGPP, PA, and DG and the phosphatase reactions involved in the metabolism of DGPP. The alpha  phosphate of DGPP is distinguished from the beta  phosphate by shading. DPP, DGPP phosphatase.
[View Larger Version of this Image (16K GIF file)]


There is a vast literature that indicates that PAP2 acts on a number of lipid phosphate compounds, which play a role in lipid signal transduction in mammalian cells (4, 7, 50). The findings presented here demonstrated that PAP2 also has a DGPP phosphatase activity. Given that this activity could regulate the levels of DGPP, PA, and DG in cells, it is clear that the role of DGPP phosphatase activity in lipid signaling as well as phospholipid metabolism should be addressed. Studies are currently in progress in our laboratories to examine the physiological roles of DGPP and DGPP phosphatase in eukaryotic cells.


FOOTNOTES

*   This work was supported by United States Public Health Service Grant GM-28140 from the National Institutes of Health (to G. M. C.) and by Grant MT 10504 from the Canadian Medical Research Council (to D. N. B.). This is New Jersey Agricultural Experiment Station Publication D-10531-1-97.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: Tel.: 908-932-9611, extension 217; Fax: 908-932-6776; E-mail: carman{at}aesop.rutgers.edu.
1   The abbreviations used are: PA, phosphatidate; DG, diacylglycerol; PAP, PA phosphatase; DGPP, diacylglycerol pyrophosphate; NEM, N-ethylmaleimide; LPA, lysophosphatidate; SPP, sphingosine 1-phosphate; CerP, ceramide 1-phosphate.
2   G. M. Zeimetz and G. M. Carman, unpublished work.

ACKNOWLEDGEMENTS

We thank Bettina Riedel and Josef Wissing for providing us with purified PA kinase and Edward A. Dennis for providing us with bromoenol lactone. We thank Jesus Balsinde and Edward A. Dennis for sharing their manuscript with us prior to publication.


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