Biochimica et Biophysica Acta 1781 (2008) 731–742

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Biochimica et Biophysica Acta

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Novel function of the human presqualene diphosphate phosphatase as a type IIphosphatidate phosphatase in phosphatidylcholine and triacylglyceridebiosynthesis pathways

Spyros Theofilopoulos, Athanasios Lykidis ⁎, George Leondaritis, Dimitra Mangoura ⁎Center for Neurosciences, Biomedical Research Foundation of the Academy of Athens, Athens 11527, Greece 1

Abbreviations: BEL, bromoenolactone; C1P, ceramphosphocholine cytidylyltransferase α; CDP-DAG, CDP-synthetase; CPT, CDP-choline:1,2-DAG choline phosphCholine; CK, choline kinase; DGAT, diglyceride acylsphocholine; LPA, lysophosphatidic acid; LPP, lipid phophosphatase/phosphotransferase; NEM, N-ethylmaleimiphosphatidic acid phosphatase; PC, phosphatidylcholinphosphatidylethanolamine; PI, phosphatidylinositosynthase; PPAPDC, phosphatidic acid phosphatase typsphingosine 1-phosphate; SM, sphingomyelin; TAG, tria⁎ Corresponding authors. A. Lykidis is to be contacted

Genome Institute, Walnut Creek, CA 94598, USA. Tel.:2965850. D.Mangoura, Center for Neurosciences, Foundathe Academy of Athens, 4 Soranou Ephesiou Street, Athe6597087/081; fax: +30 210 6597545.

E-mail addresses: alykidis@lbl.gov (A. Lykidis), mang(D. Mangoura).

1 (www.bioacademy.gr).

1388-1981/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.bbalip.2008.09.001

a b s t r a c t

a r t i c l e i n f o

Article history:

Phosphatidate phosphatase Received 8 April 2008Received in revised form 29 August 2008Accepted 11 September 2008Available online 1 October 2008

Keywords:Lipid metabolismPhosphatidate phosphatasePhosphatidic acidTricylglycerolBifunctional enzyme

s, PAPs, are key enzymes in lipid biosynthesis and signaling. Type I PAP enzymesparticipate in de-novo phospholipid biosynthesis, whereas type II PAP enzymes have an established role in lipidsignaling. To identify novel human type II PAPs potentially involved in de-novo phospholipid synthesis we usedbioinformatics to screen for enzymeswith an active site exposed to the cytosolic side ofmembranes. Two relatedenzymes, a novel lipid phosphatase related protein (LPRP-A) and a presqualene diphosphate phosphatase (PA-PSP) met this criterion. PA-PSP and LPRP-A have differential tissue and subcellular distribution, and novel yetdifferential roles in lipid metabolism. Specifically, PA-PSP, but not LPRP-A, was a potent Mg2+-independent,NEM-insensitive type II PAP. Subcellular fractionation detection indicated that both proteins were associatedwith membranes, while immunofluorescent deconvolution imaging revealed that these membranes wereexclusively from the nuclear envelope and the endoplasmic reticulum. PA-PSP overexpression, but not LPRP-A,accelerated the synthesis of phosphatidylcholine and caused accumulation of triacylglycerol with concomitantdecrease in the rate of phosphatidylinositol synthesis. Coexpression of human CTP:phosphocholinecytidylyltransferase-αwith PA-PSP enhanced the effect of PA-PSP onphosphatidylcholine levels, yet attenuatedits effect on triacylglycerol. Taken together, our studies provide the first evidence that the eukaryotic, ER-resident PA-PSP is a bifunctional enzyme with specific type II PAP activity, and regulates, in addition to type IPAPs, the de-novo biosynthesis of phospholipids and triacylglycerols.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Phosphatidic acid phosphatases (PAPs) act in concert withphosphatidate cytidylyltransferases (CDP-DAG synthetases, CDS) to

ide 1-phosphate; CCTα, CTP:diacylglycerol; CDS, CDP-DAGotransferase; CDP-Cho, CDP-transferase; GPC, glyceropho-sphate phosphatase; LPT, lipidde; PA, phosphatidic acid; PAP,e; P-Cho, Phosphocholine; PE,l; PIS, phosphatidylinositole 2 domain containing; S1P,cylglyceridesat Department of Energy Joint+1 925 2965842; fax: +1 925tion for Biomedical Research ofns 11527, Greece. Tel.: +30 210

oura@bioacademy.gr

l rights reserved.

regulate the distribution of phosphatidic acid (PA) between diacylgly-cerol (DAG) and CDP-diacylglycerol (CDP-DAG), respectively [1–4].CDP-DAG is subsequently converted to phosphatidylinositol (PI) withthe coupled activation of phosphatidylinositol synthase (PIS), whereasDAG is converted to phosphatidylcholine (PC), phosphatidylethano-lamine (PE), or triacylglycerol (TAG) with the subsequent activation ofCDP-choline:1,2-DAG choline phosphotransferase (CPT), CDP-ethano-lamine:1,2-DAG ethanolamine phosphotransferase (EPT) or diacylgly-cerol acyl transferase (DGAT), respectively [5]. In addition to their rolein lipid biosynthesis, PAP enzymes are also important in signalingcascades, as they control the balance between the phosphorylatedesters PA, lysophosphatidic acid (LPA), sphingosine-1-phosphate (S1P),and ceramide phosphate (C1P), as well as their respective depho-sphorylated derivates, namely DAG, monoacylglycerol, ceramide, andsphingosine, which are all effectors in signal transduction pathwaysevoking cell proliferation, differentiation or apoptosis [2,6,7]. In termsof biochemical properties, the PAPs that have been identified thus far ineukaryotes are functionally classified into two distinct classes: Mg2+-dependent, N-ethylmaleimide-sensitive type I PAPs (PAP1) that parti-cipate primarily in de-novo phospholipids and triacylglycerol biosyn-thesis [8–11], and Mg2+-independent, N-ethylmaleimide-insensitivetype II PAPs (PAP2), that function primarily in intracellular lipid

732 S. Theofilopoulos et al. / Biochimica et Biophysica Acta 1781 (2008) 731–742

stimulated transduction cascades [2,4,12]. Recently, lipins, the genesresponsible for fatty liver dystrophy in the fld mouse, have beenidentified as PAP1 enzymes and have been shown to be involved in de-novo phospholipid and TAG biosynthesis [11].

Mg2+-independent PAP activities have been documented for threemammalian genes, PAP2A, PAP2B and PAP2C, which display arelatively non-selective action on lipid phosphate substrates anddephosphorylate with varying specificities PA, LPA, S1P, C1P, anddiacylglycerol pyrophosphate (DAGpp) [13–17]. Because of this broadspecificity, these enzymes are often referred to as lipid phosphatephosphatases or LPP1 (PAP2A), LPP2 (PAP2C), and LPP3 (PAP2B). Allmembers are transmembrane proteins with a common active siteand identical catalytic residues [13,14,16,18]. Hydropathy analysis oftheir primary amino acid sequence suggests that their catalyticresidues are localized on the outer surface of the plasma membraneor the luminal surface of the ER membrane [19–21]. A luminalmembrane ER localization has been confirmed experimentally for therat Dri42 protein [22], a putative homolog of the human LPP3.Consequently, it has been proposed that mammalian PAP2 enzymesare not involved in de-novo phospholipid biosynthesis whichexclusively takes place on the opposite or the cytosolic surface ofthe ER membranes [23].

There is evidence, however, that members of the PAP2 family mayparticipate in de-novo phospholipids formation. For example, topolo-gical analysis of the S. cerevisiae LPP1 and diacylglycerol pyropho-sphate phosphatase 1 (DPP1), enzymes that possess Mg2+-independent PA phosphatase activity, has shown that the phosphataseactive site is located at the cytosolic side of membranes [24].Moreover, deletion of LPP1 and DPP1 in S. cerevisiae causes asubstantial decrease in the cellular levels of PI and a concomitantincrease in PA and PE levels [25,26], which strongly suggests thatthese enzymes participate in de-novo phospholipids biosynthesis.Taking these results into consideration, we initiated a bioinformaticsscreen to search for members of the PAP2 family which may directlyregulate de-novo phospholipid and TAG biosynthesis.

2. Materials and methods

2.1. Materials

[14C]-L-α-dipalmitoyl-phosphatidic acid, [14C]-glycerol, [3H]-myo-inositol and [3H]-choline were purchased from Perkin Elmer LifeSciences. [14C]-L-α-dipalmitoyl-phosphatidic acid was labelled on theglycerol moiety. L-α-palmitoyl-oleoyl-phosphatidic acid (PA), 1-oleoyl-2-hydroxy-sn-glycerol-3-phosphate (lysophosphatidic acid,LPA) and N-acetoyl-ceramide-1-phosphate (C-1-P) were purchasedfrom Avanti Polar Lipids (Alabaster, AL). Growthmedium supplementswere purchased from Gibco BRL (Grand Island, NY), restrictionendonucleases from New England Biolabs (Ipswich, MA) and DNApurification kits from Qiagen (Hilden, Germany).

2.2. RT-PCR analysis of PA-PSP and LPRP-A

Total RNA from several human cell lines and human tissue wasisolated using the RNAwiz reagent (Ambion, Austin, TX). Two μg oftotal RNA was reverse transcribed using SuperScript II reversetranscriptase (Invitrogen) and 10 ng/ml random primers. The cDNAwas amplified using Platinum Taq DNA Polymerase (Invitrogen) in thepresence of gene-specific primers. For PA-PSP, the primers utilized inanother report [48] were used: 5′-AGACCAAAGACCACCAAACG-3′ and5′-CCTACTTATCGCAGCCGTTC-3′, and for LPRP-A, the same primersthat were used for cloning the LPRP-A cDNA (see below) were utilized:5′-GGATCCACCACCATGCCAGCTTCCCAGAGCCG-3′ and 5′-GAATTCC-CAGGCAGAGATGAGCATCT-3′. For GAPDH the primers used were 5′-ATGGTGAAAGTCGGAGTCAA-3′ and 5′-ATCACAAGTTTCCCGTTCTC-3′.Band intensities were quantified by the use of ImageJ software. Levels

of transcripts in each sample were normalized with reference toGAPDHmRNA levels. Levels of GAPDH for each sample were measuredin its linear region (21 cycles). TM-31 cells were purchased from theRIKEN BioResource Center (Ibaraki, Japan), while fibroblasts were agift from Dr. Gagos (BRF), SF-268 cells from Dr. Dimas (BRF), andadipose tissue cells from Dr. Alexakis (University of Athens).

2.3. Cloning of PA-PSP and LPRP-A cDNAs and construction ofexpression vectors

Accession numbers BC038108 and BC006362 ESTs (The NIHMammalian Gene Collection, MGC, Invitrogen, Carlsbad, CA) wereused as templates for the isolation of PA-PSP and LPRP-A by PCR. Allprimers designed using the software VectorNTI, were synthesized bythe Sequencing Facility, FORTH, Crete: 5′-GGATCCACCACCATGCCAAGT-CCCCGGAGGAG-3′ and 5′-GAATTCTCGTTGACTCCACAGTAAAA-3′ forPA-PSP and 5′-GGATCCACCACCATGCCAGCTTCCCAGAGCCG-3′ and 5′-GAATTCCCAGGCAGAGATGAGCATCT-3′ for LPRP-A. BamHI restrictionsites for cloning into the expression vector pcDNA3.1-myc-His andKozak sequences to increase protein synthesis were added in thesense primers. EcoRI restriction sites for cloning into the expressionvector pcDNA3.1-myc-His were added in the antisense primers. Thestop codons on the PA-PSP and LPRP-A cDNAs were eliminated by theuse of these primers so that the myc-His oligopeptide of theexpression vector would be synthesized.

Sequence of all PCR products was routinely confirmed (MacrogenInc, Seoul, Korea). The blunt-ended PCR products generated by thePfu DNA polymerase were A-tailed using Taq DNA polymerase(Invitrogen) and dATP for 10 min, which added a 3′-terminaldeoxyadenosine to PCR fragments, were cloned into pCR II-TOPOvectors (Invitrogen), and subcloned into pcDNA3.1-myc-His vectors(Invitrogen) using the inserted BamHI and EcoRI restriction enzymesites. The pcDNA3.1 plasmid containing the CCT-alpha gene waskindly provided by Dr. Jackowski (St. Jude Children's ResearchHospital, Memphis, TN).

2.4. Cell culture and transfection

COS-7 cells were cultured in low glucose DMEM plus 10% FBSmedia, and transfected with plasmids containing PA-PSP, LPRP-A,CCTα or with the vector pcDNA3.1-myc-His using Lipofectamine 2000(Invitrogen), according to the manufacturer's instructions. In co-transfection experiments the total amount of DNA was normalizedwith empty vector.

2.5. Phosphatase enzymatic activity

The procedure described in [31] was followed with somemodifications. Briefly, [14C]-PA mixed with unlabelled PA substrate,was dried under nitrogen and resuspended by vigorous vortexing in1% Triton X-100 solution. Total cell lysate (0–50 μg) from transfectedCOS-7 cells was dissolved in assay buffer, and each assay mixturecontained final concentrations of 0.2 mM [14C]-PA (0.025 μCi/assay),0.5% Triton X-100, 100 mM Tris/maleate buffer, pH 6.5, 1 mMdithiothreitol, 0.2% albumin, 1 mM EDTA, and 1 mM EGTA (finalreaction volume of 0.1 ml). Assays were performed at 37 °C for30 min. The time of the reaction and amount of enzyme proteinadded to the reaction mixture were chosen to ensure that no morethan 15% of PA was hydrolyzed to DAG [2,14]. For PAP activityregulation, 5 μg of lysate were assayed in the presence of 3 mMMgCl2, or preincubation with 4.2 mM NEM (Sigma, Hellas) at 37 °Cfor 10 min, or, in the case of bromoenol lactone (BEL, 30 μM) orpropranolol (3 mM), the inhibitors were added in the culturemedium for 1 hour prior to harvesting. To investigate thedependence of the PA-PSP enzyme activity on the surface concen-tration of substrate, PA was presented as Triton X-100 mixed micelles

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at a surface PA concentration of 0–10 mol% [32]. The reaction wasstopped by the addition of 0.375 ml of methanol/chloroform/12 MHCl (80:40:1, v/v) and phases were separated by the addition of0.4 ml 0.1 M HCl and 0.25 ml chloroform and a 10 min centrifugationat 13,000 rpm (4 °C). The lower organic phase was collected, driedunder nitrogen, and redisolved in chloroform/methanol (2:1 v/v).The samples were flushed with nitrogen and stored at −80 °C for TLCanalysis.

2.6. Malachite green phosphate assays

Besides measuring the dephosphorylation of radiolabeled sub-strates, phosphatase activity of PA-PSP and LPRP-A was alsodetermined by the production of water soluble inorganic phosphate(Pi) using theMalachite Green Phosphate Assay kit (BioAssay Systems)as described [33,34]. Free phosphate liberated by in-vitro reactionsfrom the dephosphorylation of lysophosphatidic acid or ceramide-1-phosphate was quantitated using the malachite green reagent. Theassay mixture used was the same as the one used for measurement ofPAP activity using radiolabelled substrates (section 2.5).

2.7. Subcellular fractionation and immunoblot analysis

Forty-eight hours post-transfection, cells were harvested andhomogenates were prepared by probe sonication (7×4 sec) in a5 mM Hepes-NaOH (pH 7.2) buffer containing a cocktail of proteaseinhibitors (10 mM benzamidine, 0.3 mM PMSF, and 0.2 mMleupeptin). To obtain nuclei-, ER-, and plasma membrane-enrichedfractions, cells were homogenized using a 27 G syringe in the samelysis buffer and the homogenates were centrifuged at 500×g for 5 minat 4 °C; low speed supernatants were then centrifuged at 1000×g for10 min, followed by 20,000×g for 20 min, and 100,000×g for 2 h at4 °C. Each of the pellets were resuspended in five volumes ofhomogenization buffer. Proteins from total cell lysate or subcellularfractions, normalized for protein content were analyzed with SDS-PAGE in 12% gels. Twenty μg of proteinwas loaded in eachwell and theresolved proteins were transferred onto nitrocellulose membranes asdescribed [27]. Abundance and electrophoretic mobility of PA-PSP,LPRP-A, lamin or calnexin were detected by blotting the membraneswith the relevant antibody (mouse monoclonal c-Myc 9E10; mousemonoclonal lamin A/C, or goat polyclonal calnexin C-20, all from SantaCruz Biotechnology, Santa Cruz, CA), and then with the appropriatespecies-specific secondary antibodies conjugated to horseradishperoxidase (HRP) or to alkaline phosphatase (AP) [27,28]. Immunor-eactivity was visualized by further incubation with ECL chemilumi-nescence (Pierce, Rockford, IL) or with the AP Substrate kit (Biorad,Hercules, CA).

2.8. Immunocytochemistry

PA-PSP- or LPRP-A- or vector-transfected COS-7 cells were grownon glass coverslips. Cells were first washed with PBS containing CaCl2and MgCl2, fixed in 4% parafolmadehyde for 25 min at roomtemperature and permeabilized with 0.1% Triton X-100 in PBS[28,29]. After blocking non-specific binding with 3% normal goat ordonkey serum in PBS, PA-PSP or LPRP-Awere detected with mAb to c-Myc 9E10, rabbit polyclonal to His (Cell Signaling, Danvers, MA), goatpolyclonal to calnexin, rabbit polyclonal to beta-COP (AffinityBioreagents, Golden, CO), or mAb to lamin A/C antibodies; secondaryantibodies were goat-anti-mouse-rhodamine and goat-anti-rabbit-FITC, or donkey anti-mouse FITC and donkey anti-goat-rhodamine.Nuclear DNAwas stained with Hoechst 33258 (Pharmigen, San Diego,CA). GFP-tagged cannabinoid type-1 (CB1) receptor plasmid was akind gift by Dr. Lenkei (Ecole Supérieure de Physique et de ChimieIndustrielles, Paris) [30]. Cellular staining was visualized using anAxiovert 200 M inverted fluorescence microscope with a motorized

stage, equipped with a 63x oil lens and a Hamamatsu Orca-ER CCDcamera. Images from triple or double staining were obtained at thesame focal planes of serial 0.5 μm-thick Z dimension optical sectionsand resulting Z-stacks were deconvoluted with the nearest neighboralgorithm using the Slidebook™ software (Imaging facility, Center forNeurosciences, BRF). Quantitative fluorescent measurements wereperformed as described [28].

2.9. Labeling, isolation, and quantitation of lipids

Twenty four hours post-transfection, COS-7 cells were grown inmedia containing [3H]-inositol, [3H]-choline (3 μCi/ml medium), or[14C]-glycerol (1 μCi/ml medium) for 24 h and then harvested foranalysis. For short-term labeling experiments, transfected cells werefirst washed three times with PBS and pre-incubated for 1 h withDMEM containing 0.5% fatty acid-free BSA and 100 μM palmitic acid;the latter was solubilized by vigorous vortexing in DMEM containing3% fatty acid-free BSA and then diluted with fresh DMEM. Cells weresubsequently incubated with [3H]-choline or [3H]-inositol, 4 and8 μCi/ml medium, respectively, for 1–6 h. Cells were washed twicewith PBS, harvested and lysed in 1.5 ml 0.5 M ice cold perchloric acid(15 min with frequent vortexing). Cell lysates were centrifuged at13,000 rpm for 10 min at 4 °C and the supernatant was collected foranalysis of inositol/choline/glycerol uptake and for quantitation ofphosphocholine (P-Cho), CDP-Choline (CDP-Cho), or glycerophos-phocholine (GPC) production. Cell pellets were washed with 1 mlperchloric acid containing 1 mM EDTA, and then with 1 ml 1 mMEDTA pH 6.0. Lipids were extracted by vortexing vigorously the pelletin 0.75 ml methanol/chloroform/12 MHCl (80:40:1) [35]. Phases wereseparated as described above.

TAG and DAG were resolved from phospholipids by TLC in hexane/diethyl ether/acetic acid 80:20:2 v/v along with individual lipidstandards. Different phospholipids (PI, PC, PE, PS, SM, cardiolipin)were resolved from TAG-DAG by TLC in chloroform/methanol/ammonium hydroxide 60:35:8 v/v. Choline, glycerol, P-Cho, CDP-Cho and GPC were resolved by TLC in 2% ammonium hydroxide/95%ethanol (1:1, v/v). In the case of the in-vitro phosphatase assays, PAand DAG were separated by TLC in hexane/acetic acid/water 70:30:1v/v. The organic phase was separated on Silica gel 60 F254 TLC plates(20×20 cm, Merck, Whitehouse Station, NJ), whereas supernatantswere run on Silica gel G (20×20 cm, Uniplate, Tamworth, UK).Radioactivity was measured using a TLC Bioscan AR-2000 ImagingScanner and the WinScan 3 software for analysis (Center forNeurosciences Core Facility, BRF). The specific radioactivities of theprecursors, taking into account the inositol and choline that wascontained in the culturing medium, were 0.075 μCi/nmol for inositol,0.112 μCi/nmol for choline, and 0.143 μCi/nmol for glycerol.

In addition to the TLC scanner analysis, radioactivity of each lipidwas measured by liquid scintillation, as described [36]. Briefly, bandsvisualized after exposure to iodine vapors and identified by comparisonwith commercially bought standards, were scraped for radioactivitydetermination using a Win Spectral α/β, 1414 Liquid Scintillationcounter (Perkin Elmer Life Sciences, Boston, MA). Data were analyzedusing the software Wallac LSC.

2.10. Detection of total cellular cholesterol and TAG levels in cultures

Total cellular levels of cholesterol and triglycerides in COS-7 cellcultures, transfected with PA-PSP or LPRP-A, was measured after lipidextraction, using a ChemWell 2910 Automated EIA and ChemistryAnalyzer (Awareness Technology Inc, Palm City, Florida; Centre forExperimental Surgery, BRF). For cholesterol, the detection system ofcholesterol oxidase/peroxidase/phenol/4-aminoantipyrine was used;whereas for triacylglycerides, glycerol phosphate oxidase (GPO) wasused instead of cholesterol oxidase. The red colored quinonimine dyeproduced was read at 500 nm.

Fig. 1. Differential expression levels of message for PA-PSP and LPRP-A in human cells.Representative images of semi-quantitative RT–PCR analyses show levels of PA-PSP,LPRP-A, and GAPDH transcripts in human cell lines and normal human cells.

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2.11. Statistics

All experiments were performed three or more times and datawere analyzed by ANOVA and the Student Newman-Keuls Post-HocANOVA test.

3. Results

3.1. Identification of putative human PAP2 enzymes with cytosolic activesites and expression profiles in human cells

In order to identify mammalian genes with potential PAP2/LPPactivity, we created Hidden Markov statistical models (HMMs; http://hmmer.wustl.edu) for the conserved catalytic C1-C2-C3 domainsequences of all known type II PAPs (PAP2s) [7]. HMM searches inthe human genome pulled out all known genes for the LPT (lipidphosphatase/ phosphotransferase) family of proteins [2,7]: three lipidphosphate phosphatases (PAP2A/LPP1, PAP2B/LPP3, PAP2C/LPP2) [14–16,37]; four lipid phosphatase-related proteins/plasticity relatedgenes (LPR/PRGs) [38,39]; two sphingosine phosphate phosphatases(SPPs) [40–42]; two sphingomyelin synthetases (SMSs) together withthe uncharacterized sphingomyelin synthetase related gene [43], thedolichyl-pyrophosphate phosphatase (DLPP) [44]; the recently iden-tified PPAPDC1B (phosphatidic acid phosphatase type 2 domaincontaining 1B; also known as HTPAP) [45] and PAP2D genes [46];and three more potential PAPs, which we originally designated asLipid Phosphatase Related Proteins LPRP-A (Genbank accessionnumber gi|33871607, on human chromosome 9, q34.13), LPRP-B(Genbank accession number gi|66773040, on human chromosome 9,p24.1), and LPRP-C (Genbank accession number gi|73611920, onhuman chromosome 10, q26.12). The latter was very recentlydescribed as a diacylglycerol pyrophosphate phosphatase-like protein,DPPL1 [47].

We then applied a second bioinformatics screen, in order todistinguish which enzyme among LPRP-A, B, and C may be involved inde-novo phospholipid metabolism. Newly synthesized PA is typicallyformed on the cytosolic side of the ER membranes and therefore weexamined whether these candidates possibly possessed proteinstructures compatible with such localization. Using TopPred softwareformembrane proteins and the Kyte Doolittle scale as well as TMHMMthat predict both localization and orientation of transmembranehelices and hence a cytosolic versus a luminal localization of the inter-helices regions (intervening loop regions), we analyzed membraneenzymes involved in phospholipid metabolism with known topology.Specifically we analyzed thirty five enzymes from the CDS, PAP andphosphatidyltransferase families, including human CPT, CEPT, PIS,CDS1, sphingomyelin synthetase, LPP1, E. coli phosphatidylglycerolphosphate synthetase and CDS. For each enzyme, the softwarepredicted the correct topology for both prokaryotic or eukaryoticproteins. Because this topology analysis predicted that the active siteof PPAPDC1B, PAP2D, and DPPL1 would be localized on the luminalside of intracellular compartments or the extracellular surface of theplasma membrane, we did not study further DPPL1. The same analysispredicted that the active site of LPRP-A and B were localized on thecytosolic side of a membrane. Thus far all characterized human LPPenzymes expose their active site to the exterior of the plasmamembrane or the lumen of intracellular organelles, and therefore thisnovelty could possibly indicate that LPRP-A and B have access to newlyformed PA. Because LPRP-B was identical to the recently identifiedpresqualene diphosphate phosphatase (PSP) which dephosphorylatespresqualene diphosphate, an intermediate in squalene formation anda precursor of cholesterol [48], we redesignated LPRP-B as PA-PSP.

We first examined expression levels of PA-PSP and LPRP-A inseveral cell types of human origin using semi-quantitative RT-PCRanalysis in RNA isolated from cell lines (TM-31 astrocytoma, SF-268glioblastoma, cervical epithelial HeLa, embryonic kidney HEK-293,

prostate cancer PC-3, breast cancer MCF-7) or normal tissue(amniocytes, fibroblasts, and adipose tissue). The expression profilesof PA-PSP and LPRP-A differed significantly between these cells (Fig.1A). PA-PSP expressionwas relatively high in HEK, fibroblast, and TM-31 cells, whereas LPRP-A expression was approximately 3-fold higherthan PA-PSP in TM-31 cells and very low in all other cell types.Interestingly, neither enzyme was expressed in human adipose tissuecells.

3.2. PAP Type II enzymatic properties of PA-PSP

PA-PSP and LPRP-A shared a 44.3% identity and 60.3% similarity intheir amino acid sequences and, compared to LPP1, LPP2 and LPP3,possessed an extended NH2 terminus that preceded the firsttransmembrane domain. Both were predicted by the TopPred andTMHMM topology analysis to possess six transmembrane α-helices.PA-PSP had entirely conserved the active site phosphatase motifsequence of the LPT family of proteins [7], namely C1: KXXXXXXRP,C2: SGH and C3: SRXXXXXHXXXD, whereas LPRP-A had a variant C1motif, with a glutamine (residue 163 on LPRP-A) replacing a conservedlysine and a glycine (residue 170 on LPRP-A) replacing a conservedarginine. Each of the two identified human genes was amplified byPCR, cloned, and subcloned in the pcDNA3.1-myc-His mammalianexpression vector, as described in Materials and methods. COS-7 cellswere transfected with pcDNA3.1-myc-His-PA-PSP or pcDNA3.1-myc-His-LPRP-A plasmids and lysates were routinely analyzed by SDS-PAGE and Western blotting with a mAb to myc 48 h later.

PAP activity of each enzyme was assessed in total lysate from cellsoverexpressing PA-PSP and LPRP-A using [14C]-PA as a substrate andmeasuring formation of [14C]-DAG (Fig. 2). Low endogenous PAPactivity was detected in vector-transfected cell lysates with a specificactivity of 0.476 nmol/min/mg. Cells expressing LPRP-A exhibited asmall, 1.7-fold increase in PAP activity, or a specific activity of0.804 nmol/min/mg. On the contrary, cells expressing PA-PSPexhibited a 13-fold increase reaching a specific activity of6.232 nmol/min/mg. The activity of the PA-PSP and LPRP-A enzymeswas linear with respect to protein amount (0-50 μg of total cell lysate,Fig. 2A) and reaction time (0-60 min, not shown).

We next examined the dependency of the activity of PA-PSP onMg2+, NEM and possible inhibition by the established PAP inhibitorsbromoenolactone (BEL) [49,50] and propranolol [3,13,50]. Neither thepresence of 3 mM MgCl2 in the assay buffer nor pre-incubation of theprotein with NEM altered the levels of DAG formed (Fig. 2B).Moreover, the phosphatase enzymatic activity of PA-PSP was notinhibited by BEL, but preincubation of cell cultures with propranololresulted in a significant 75% reduction in PAP activity (Fig. 2B). Takingtogether that the enzymatic activity of PA-PSP was independent ofMg2+, insensitive to NEM or BEL treatment, and inhibited by

Fig. 3. Biochemical fractionation of PA-PSP and LPRP-A. To obtain nuclear or ERmembrane-enriched fractions, PA-PSP- or LPRP-A-transfected COS-7 cells weremechanically homogenized in detergent-free lysis buffer and the homogenates werecentrifuged at 500×g for 5 min (P500); from then on each supernatant was sequentiallycentrifuged at 1000×g for 10min (P1000), 20,000×g for 20min (P20,000), and finally at100,000×g for 2 h to yield a final pellet (P100,000) and membrane free cytosol(S100,000). Every pellet was resuspended in five volumes of homogenization buffer and20 μg of protein was loaded in each well of a 12% SDS-PAGE gel. The resolved proteinswere transferred onto nitrocellulose membranes, and abundance of PA-PSP and LPRP-A,lamin, or calnexin in PA-PSP- or LPRP-A-transfected cells was detected by Westernblotting with mouse mAbs against c-Myc, lamin A/C, or a goat pAb to calnexin,respectively. Right arrows point to the overexpressed PA-PSP and LPRP-A. Left arrowsindicate the position of prestained Mr markers.

Fig. 2. Catalytic PAP2 activity of PA-PSP but not LPRP-A. (A) Indicated protein amounts of total lysate from COS-7 cells expressing PA-PSP or LPRP-A were assayed for PAP activity asdescribed in the Materials and methods section. (B) PA-PSP-expressing cells or cell lysates were treated with MgCl2, NEM, BEL and propranolol as described in the Materials andmethods section and assayed for PAP activity. Data are mean values±S.E.M. of three separate experiments. Asterisks indicate statistically significant difference (Pb0.01) compared to−MgCl2. (C) 5 μg of total cell lysate from cells expressing PA-PSP were assayed for PAP activity at the indicated surface concentrations (mol %) of PA. (D) C1P (upper panel) and LPA(lower panel) were tested as substrates for PA-PSP and LPRP-A using the Malachite Green Phosphate method.

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propranolol, our data are consistent with the classification of theenzyme as a type II phosphatase.

To explore the basic kinetic properties of PA-PSP, we determined itsactivity using Triton X-100/PA mixed micelles as a provider of surfacefor catalysis [32]. Analysis of the kinetic data parameters of the PA-PSPprotein showed that PA-PSP exhibited a Vmax value of 44.8 nmol/min/mg and a Km value for PA of 4.9 mol% (Fig. 2C).

We also utilized lysophosphatidic acid (LPA) and ceramide-1-phosphate (C1P) as potential substrates of PA-PSP and LPRP-A. In cellsoverexpressing PA-PSP, there was a 40% increase in the amount ofdephosphorylated C1P compared to vehicle-transfected cells (Fig. 2D,upper panel). However, there was no difference in LPRP-A-over-expressing cells. Therefore, PA-PSP, unlike LPRP-A, can also depho-sphorylate ceramide 1-phosphate, besides PA. Neither PA-PSP norLPRP-A could dephosphorylate LPA, suggesting that LPA cannot beused as a substrate by any of the two proteins (Fig. 2D, lower panel).

3.3. Subcellular localization of PA-PSP and LPRP-A

To investigate the subcellular localization of PA-PSP and LPRP-A,proteins from subcellular fractions from transfected COS-7 cells wereanalyzed by SDS-PAGE and Western blotting with a mouse antibodyagainst the myc epitope. Both PA-PSP and LPRP-A were expressed atthe expected Mr of 32.4 kDa and 29.5 kDa, each increased by 4.7 kDa

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Fig. 5. Overview of phospholipid and triacylglycerol biosynthetic pathways. De-novoformed PA is distributed between CDP-DAG and DAG. DAG is subsequently directed toPC via the CDP-choline pathway. Saturation of the CDP-choline pathway causesdiversion of excess DAG towards TAG. PIS, phosphatidylinositol synthase; CCT, CTP:choline-phosphate cytidyltransferase; CDS, CDP-DAG synthetase; CK, choline kinase;CPT, CDPcholine:1,2-DAG choline phosphotransferase;DGAT, diglycerol acyl transferase;PLAs, Phospholipases A.

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from the addition of the myc-His epitopes, that is 37.1 kDa and34.2 kDa, respectively (Fig. 3, upper panel). In accordance with thehydropathy analysis that the six transmembrane α-helices of eachprotein would primarily target them to membranes, both enzymeswere detected exclusively in membrane fractions. First, PA-PSP andLPRP-A were detected in the 500×g pellet of detergent-free total celllysate (P500 lanes) which consists primarily of intact nuclei and someunbroken cells; further centrifugation of the supernatant at 1000×grecovered most of the proteins at the pellet (P1000 lanes), a nuclearmembrane-rich fraction (Fig. 3, upper panel). Subsequent centrifuga-tion of the P1000 supernatant at 20,000×g generated a pellet enrichedin membranes from ER, mitochondria, and lysosomes (P20,000 lanes),where both PA-PSP and LPRP-A proteins were detected. Finally, bothproteins were present to a lesser extent, in the 100,000×g pellet of the20,000×g supernatant which mainly contains microsomal mem-branes (P100,000 lanes) and clearly absent in the organelle-freecytosol fraction (S100,000 lanes). Western blotting for the abundanceof the intermediate filament protein lamin in the same fractions(Fig. 3, middle panel) verified the presence of nuclear envelopes inP500 and P1000 fractions and minimal contamination of the rest ofthe fractions. Similar analysis for the ER membrane protein calnexin(Fig. 3, lower panel) indicated that ER membranes from the variousstructures of ER were found in all the pellets and in particular in the100,000×g pellet where smaller diameter structures reside. Laminand calnexin were not detected in the cytosol further validating theobservation that PA-PSP and LPRP-A were not soluble, but tightly-associated with the membrane proteins.

The specific localization of PA-PSP and LPRP-A in endomembraneswas further studied at the single cell level in transfected cells withanalysis of their immunofluorescence staining patterns using an anti-myc monoclonal or anti-his polyclonal antibody. The localization ofPA-PSP or LPRP was studied relatively to immunostaining patterns ofmembrane markers, namely of calnexin to identify ER membranes,beta-COP for Golgi network, or lamin A/C for nuclear envelope, allcounterstained for nuclear DNA with Hoechst 33258 (Fig. 4), as

Fig. 4. Close association of PA-PSP and LPRP-A with the nuclear envelope and ER structures.anti-myc monoclonal or anti-His polyclonal antibodies and appropriate FITC (green) or rhodstained with antibodies against endogenous calnexin (B, J, and merged images), lamin (D, Lplasma membrane association of PA-PSP or LPRP-A, cells were co-transfected with PA-PSP or33258 (DAPI). Bar=10 μm.

described in Section 2.8. PA-PSP or LPRP-A staining heavily decoratedthe perinuclear region, arranged as a ring in close association withthe nuclear envelope with a radiating network of branching tubuleswhich is typical of ER structures. Indeed, a significant co-localizationwas observed with the ER marker calnexin in the nuclear envelopeand peripheral ER stacked cisternae and tubules (arrows in Fig. 4A, Band I, J and merged images). As estimated by quantitative immuno-fluorescence, measurements, co-localization of calnexin with LPRP-Awas N90%, compared to 50% for PA-PSP. Similarly, co-localization ofPA-PSP or LPRP-A with lamin in the nuclear envelope membraneswas also considerable (arrows in Fig. 4C, D and K, L and mergedimages, respectively). On the contrary, when Golgi membranes werestained with anti-beta-COP antibodies, no significant overlap wasobserved (Fig. 4E, F and M, N and merged image for PA-PSP and LPRP,respectively). The putative PAP enzymes were not detected at theplasma membrane either, as no overlap was seen when cells wereco-transfected with the GFP-tagged, plasma membrane boundcannabinoid type-1 (CB1) receptor (Fig. 4G, H and O, P and mergedimages).

3.4. PA-PSP regulates phospholipid and triacylglycerol biosynthesis

The main metabolic pathways that phosphatidic acid phosphataseenzymes are implicated in is depicted in Fig. 5. It has previously beenshown that the primary cellular destination of DAG is PC andconsequently the CDP-choline pathway controls short-term TAGformation [51]. The CDP-choline pathway is the major consumer ofnewly formed DAG and it is only after its saturation that excess DAG isdiverted towards TAG. To investigate the effect of the PA-PSP enzymein de-novo lipid biosynthesis, we overexpressed PA-PSP in COS-7 cells,labeled the cells with the appropriate precursor, namely glycerol,choline, or inositol, and monitored the incorporation of the label intospecific lipid species. LPRP-A was included in all these experiments asan additional control, thus ensuring that changes in lipid metabolismwere specific and not simply a non-physiological result from theectopic overexpression of proteins. With all isotopes studied, therewas no difference in the total incorporation of any radiolabeledprecursor into lipid- and water-soluble radioactivity among vector-,PA-PSP-, or LPRP-A-transfected cultures.

We then measured incorporation of [14C]-glycerol into specificphospholipids and TAG. Overexpression of PA-PSP increased theincorporation of [14C]-glycerol into TAG and PC by a significant 8-foldcompared to control (from 3±1 dpm/μg protein to 25±1 dpm/μg, Fig.6A) and 25% (from 66±3 dpm/μg to 81±10 dpm/μg, Fig. 6A),respectively. Concomitantly, PA-PSP overexpression resulted in asmall but statistically significant 17% decrease in the incorporationof label into [14C]-PI (from 11±0 dpm/μg to 9±0 dpm/μg, Fig. 6A). Onthe contrary overexpression of LPRP-A did not affect the incorporationof label into either phospholipids or TAG (Fig. 6A). These dataindicated that PA-PSP enzyme levels modulate the distribution ofglycerol between phospholipid species and TAG. To further demon-strate that PA-PSP levels affect the de-novo pathway of phospholipidbiosynthesis, we transfected COS-7 cells with PA-PSP, LPRP-A, orcontrol vector and we labeled the cells with either [3H]-choline or[3H]-inositol. Overexpression of PA-PSP resulted in a significant 23%increase in the incorporation of choline into [3H]-PC (from 3701±174 dpm/μg protein to 4556±205 dpm/μg, Fig. 6B) accompanied by a57% decrease in the incorporation of [3H]-inositol into PI (from 56±4 dpm/μg protein to 36±3 dpm/μg, Fig. 6C). On the contraryoverexpression of LPRP-A had no effect on either PC or PI (Fig. 6B, C).

PA-PSP (A–H and merged images) or LPRP-A (I-S and merged images) were detected byamine (red) conjugated secondary antibodies as indicated in each panel. Cells were co-, and merged images), and beta-COP (F, N, and merged images). In order to detect anyLPRP-A and CB1-GFP plasmids (H, P). In all cases, nuclear DNAwas stained with Hoechst

Fig. 6. PA-PSP but not LPRP-A regulates de-novo lipid biosynthesis. COS-7 cells expressing PA-PSP or LPRP-Awere labeled with (A) [14C]-glycerol, (B) [3H]-choline, (C) [3H]-inositol for24 h, or (D) PA-PSP-expressing cells were pre-incubated with palmitic acid (100 μM)-BSA complexes 1 h before the addition of [3H]-choline or [3H]-inositol, for the indicated times. Inall cases, lipids were extracted and separated by TLC as described in the Materials and methods section. Individual [14C]- or [3H]-labeled lipid molecules were identified according tostandards andwere quantitated using a TLC scanner and liquid scintillation. PA-PSP, but not LPRP-A, resulted in an increase of TAG and PC levels, and a decrease in PI levels. PE and SMlevels remained unchanged. Data are mean values±S.E.M. of three separate experiments. Asterisks indicate statistically significant difference (Pb0.01) compared to control.

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Neither PA-PSP nor LPRP-A had a detectable effect on the incor-poration of [14C]-glycerol into [14C]-PE (Fig. 6A), or of [3H]-cholineinto [3H]-sphingomyelin (SM) (Fig. 6B and C).

We followed the incorporation of radiolabeled choline and inositolduring short-term labeling in order to ensure that the observeddifferences were due to changes in the rates of lipid synthesis and notlipid head-group turnover. Furthermore, in order to enhance the effectof PA-PSP, we also stimulated glycerophospholipid synthesis usingpalmitic acid complexed to BSA, so that synthesis would not dependon phospholipid turnover for fatty acids supply. In pilot experiments,we established that this manipulation enhanced the incorporation of[3H]-choline into [3H]-PC, in vector-transfected cells, at least 2-foldover a time period of 4 h. Overexpression of PA-PSP resulted inprogressively increased rates of incorporation of [3H]-choline into[3H]-PC, reaching a 17 % difference over vector-transfected cells by 4 hof labeling (Fig. 6D, upper panel). [3H]-inositol incorporation into [3H]-

PI was generally found to be reduced, the differences, however, werenot stastistically significant compared to vector-transfected cells,throughout the labeling time period of 2–4 h (Fig. 6D, lower panel).These results further support our conclusion from the long-termlabeling studies that PA-PSP affected the de-novo biosynthetic path-way of phospholipids and particularly that of PC by channelingdiacylglycerol into the CDP-choline pathway.

Finally, quantitation of total cellular levels of TAG and cholesterolin vector- or PA-PSP- or LPRP-A-transfected cells, revealed a 1.55 foldincrease in the amount of TAG in PA-PSP-transfected cells as comparedto vector-transfected cells (data not shown). LPRP-A-transfected cellsshowed no significant difference compared to control. Also, there wasno significant difference in the total cellular cholesterol levelsbetween control, PA-PSP- or LPRP-A-transfected cells (data notshown) which showed that PA-PSP or LPRP-A had no effect on totalcellular cholesterol levels.

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3.5. PA-PSP activity is functionally coupled to CCTα

To further examine the contribution of PA-PSP in de-novophospholipid biosynthesis we overexpressed PA-PSP alone or incombination with human CTP:phosphocholine cytidyltransferase α(CCTα, a key regulator of PC biosynthesis) [52] andwe labeled the cellswith either [14C]-glycerol or [3H]-choline. Consistent with ourprevious observations PA-PSP increased the incorporation of [14C]-glycerol into TAG, whereas overexpression of CCTα alone had nostatistically significant effect (Fig. 7A). However, co-expression ofCCTα and PA-PSP resulted in attenuation of the PA-PSP effect on TAG.The incorporation of glycerol into TAG in the presence of CCTα wasonly 50% of the one observed when only PA-PSP was overexpressed(Fig. 7A). Importantly, overexpression of CCTα resulted in theredirection of the excess DAG produced from PA-PSP towards PCand away from TAG. Indeed, PA-PSP or CCTα increased incorporationof [14C]-glycerol into PC by 28% and 37% respectively as compared tocontrol, whereas co-expression of both had an additive effect, andglycerol incorporation into PC reached almost a 2-fold (86%) increasecompared to control (Fig. 7B). Similar increases in PC synthesis wereobtained when [3H]-choline was used as the labeled precursor (Fig.7C). In parallel, we examined the incorporation of [3H]-choline intothe water-soluble intermediates and metabolites of the CDP-cholinepathway [3], i.e. phosphocholine (P-Cho), CDP-Choline (CDP-Cho) andglycerophosphocholine (GPC), in the cytosol of cells overexpressingPA-PSP, CCTα or both. The excess of PC generated by PA-PSP and/orCCTα was converted to GPC, consistent with the excess phospholipidresponse reported in previous investigations [53] (Fig. 7D). Also, PA-

Fig. 7. CCTα overexpression attenuates the lipogenic effect of PA-PSP and enhances PC formatior [3H]-choline (C), as described in the Materials and methods section. Lipids were extractedaccording to standards and were quantitated using a TLC scanner and liquid scintillation.moleculeswere separated by TLC. [3H]-labeled P-Cho, CDP-Cho andGPCwere identified accorof three separate experiments. Asterisks indicate statistically significant difference (Pb0.01)

PSP, unlike CCTα, did not result in increase of P-Cho and CDP-Cholevels (Fig. 7D), which showed that PA-PSP was not involved in theregulation of the CDP-Cho biosynthetic pathway. These data stronglysuggested that PA-PSP and CCTα act in synchrony to regulate cellularlipid metabolism.

4. Discussion

The hydrolysis of de-novo formed PA to DAG is a major branchingpoint regulating the distribution and diversity of lipid species. Untilnow, only the type I PAP has been implicated in the de-novobiosynthesis of phospholipids and TAG [11]. Our results show thatthe PA-PSP enzyme with known presqualene diphosphate phospha-tase activity [48] participates in addition in de-novo phospholipid andneutral lipid biosynthesis.

The biochemical characterization of the PA-PSP and LPRP-Aenzymes showed that PA-PSP, but not LPRP-A, is a Mg2+-independentand NEM-insensitive PAP. Furthermore, its phosphatase activity isinhibited by propranolol, but not by BEL. LPRP-A has a variant C1domain in which a conserved lysine and arginine are not present.According to the established active site configuration and reactionchemistry of the vanadium-dependent chloroperoxidase (CPO), whichshares a similar reaction mechanism with PAPs [7], the two missingresidues are essential for phosphatase activity, and our resultsshowing lack of PAP activity for LPRP-A support this model.

PA-PSP and LPRP-A are clearly evolutionary related genes, yet, theyappear to have distinct enzymological properties and may servedifferent cellular functions. PA-PSP but not LPRP-A enzyme levels had

on. COS-7 cells expressing PA-PSP and/or CCTαwere labeledwith [14C]-glycerol (A and B),and separated by TLC. Individual [14C]- or [3H]-labeled lipid molecules were identified

(D) the supernatant from the [3H]-choline experiment was collected for analysis, andding to standards andwere quantitated using a TLC scanner. Data aremean values±S.E.M.compared to control.

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profound effects on de-novo lipid biosynthesis. Overexpression of PA-PSP in COS-7 cells led to acceleration of PC biosynthesis with aconcomitant accumulation of TAG and deceleration of PI biosynthesis;PE and SM levels remained unchanged. On the contrary, LPRP-A hadno effect on lipid biosynthesis. These data support the conclusion thatPA-PSP may regulate the flux of newly synthesized PA towards PC andTAG.

Co-expression of CCTα with PA-PSP enhanced the effect of PA-PSPon PC and attenuated the effect of PA-PSP on TAG. Moreover, we foundthat PA-PSP was not involved in the regulation of the CDP-Chobiosynthetic pathway but worked in synchrony with CCTα to regulatecellular lipid metabolism. In addition, PA-PSP increased total cellularlevels of TAG by 1.55 fold compared to control cultures. This suggeststhat the TAG formed by PA-PSP is only a small proportion compared tothe total TAG mass in the cells, hence the difference in the increasesbetween radiolabeled TAG and total cellular TAG.

PA-PSP and LPRP-A have been assigned, during the course of thiswork, by another bioinformatics analysis as PPAPDC2 (phosphatidicacid phosphatase type 2 domain containing 2) and PPAPDC3(phosphatidic acid phosphatase type 2 domain containing 3)respectively. PA-PSP and LPRP-A have been also provisionally labeledas candidate sphingomyelin synthase type 2β (CSS2β, SwissProt/TrEMBL accession number Q8IY26) and type 2α (CSS2α, SwissProt/TrEMBL accession number Q96SS7) respectively, because of sequencehomology with sphingomyelin synthases [7,43]. CCSβ has not beentested for sphingomyelin synthase activity, whereas CCS2α wasexpressed in S. cerevisiae and was found not to exhibit sphingomyelinsynthase activity [43]. As stated earlier, when expressed as a His-fusion protein, membrane-bound PA-PSP exhibits in-vitro presqualenediphosphate phosphatase activity [48]. Presqualene diphosphate is anintermediate in squalene formation and a precursor of cholesterol.Other members of the LPT family, for example the well characterizedhuman LPP1, LPP2 and LPP3 also exhibit differential substratespecificity for LPA, C1P, S1P and PA [4,16,20,54]. When we used LPAand C1P as potential substrates for PA-PSP and LPRP-A we found thatPA-PSP, unlike LPRP-A, could also dephosphorylate ceramide 1-phosphate but not lysophosphatidate. Collectively, these data suggesta wider diversion in substrate specificity for PA-PSP as compared tothe well-characterized LPP1, LPP2 and LPP3. Thus, PA-PSP may be abifunctional PAP2 that coordinates fluxes towards both de-novophospholipid/triglyceride metabolism and away from squalene/cholesterol synthesis.

This possibility is further supported by phylogenetic analysis data(not shown), which indicate that unicellular organisms, like D.discoideum or S. cerevisiae, do not possess any PA-PSP orthologs. Anortholog of the PA-PSP subfamily appears first in the multicellular D.melanogaster, C. elegans and A. thaliana. D. melanogaster and C. elegansdo not possess squalene synthetase neither de-novo cholesterolbiosynthesis but possess orthologs of PA-PSP. Therefore it appearsthat the primal and earliest role of PA-PSP is in the de-novophospholipids metabolism, and future structure studies may possiblyexplain how PA-PSP acquired additional enzymatic roles in highermulticellular organisms.

In terms of subcellular localization human LPPs have been shownto exhibit cell-specific localization to detergent-resistant membranedomains [55,56], where they may be compartmentalized with LPA orS1P receptors or with lipid signaling enzymes, particularly phospho-lipase D. LPP1 and LPP3, specifically, have been shown to co-localizewith PLD2 in caveolin-enriched detergent-resistant microdomains,where they metabolize PLD2-derived PA [56]. Our fractionation andimmunocytochemistry experiments revealed that PA-PSP and LPRP-Aassociated with the ER, where they co-localize with calnexin, anintegral ER resident protein, as well as with the nuclear envelope, acontinuoum of the rough ER membranes, where they co-localize withlamin A/C. As such, a significant part of PA-PSP and LPRP-A, at leastwhen expressed in COS-7 cells, is associated with low speed pellet,

which probably consists of nuclear and ER membranes. It is likely thatPA-PSP or LPRP-A are integral components of the nuclear envelope,since, under the most favorable conditions, we did not detect anyintranuclear immunoreactivity of either proteins. The latter has alsobeen suggested by a subtractive proteomic screen in liver cells [57].This is in accordance with previous studies that have identifiedenzyme activities generating DAG from PA in nuclei [7,58,59].Therefore PA-PSP is likely to be a candidate PAP enzyme that canregulate nuclear PA/DAG pools.

In addition to their perinuclear localization, a significant co-localization of PA-PSP or LPRP-A was also observed with calnexin inperipheral ER structures/tubules. Moreover, when cells expressedhigh levels of LPRP-A we observed the induction of an ER phenotype(data not shown), similar to that established for ER resident proteins,such as cytochrome b [60]. This phenotype was consistent with there-distribution of calnexin-rich ER membranes to lower-speed pelletsobserved after subcellular fractionation analysis in the case of LPRP-A(Fig. 3). Elevated levels of such proteins function to regulate theformation of stacked ER membrane arrays (termed organized smoothER or OSER) through weak homotypic interactions between cyto-plasmic domains of membrane proteins on opposing membranes[60]. A role of LPRP-A as an ER resident protein involved in ERdifferentiation, a dynamic phenomenon which participates in cellproliferation and differentiation remains, however, to be determined.Overall, our analysis of the subcellular localization of PA-PSP alongwith the prediction studies on the topology of the PA-PSP active siteprovide a cellular basis for the role that the biochemical analysis hassuggested for PA-PSP as an enzyme that may regulate de-novo lipidmetabolism.

PA-PSP and LPRP-A message expression differed greatly amonghuman cell types as well as when compared to each other. This is inagreement with EST (UniGene's ProfileViewer) analysis where PA-PSPand LPRP-A display a wide tissue distribution, with PA-PSP having ahigher expression in the human bladder, larynx, thymus and pharynx(ESTProfileViewer, Hs.107510) and LPRP-A in the heart, muscle, brainand tongue (ESTProfileViewer, Hs.134292). It is interesting that in TM-31 cells, an astrocytoma cell line heterozygous for the neurofibrominprotein and p53, LPRP-A expression levels were 3-fold higher than innormal glioblastoma SF-268 cells. This may be an indication thatLPRP-A expression is regulated by the Ras signaling pathway. Moreinteresting is, however, the fact that the adipose tissue is null foreither PAP2s, which may suggest that at least the action of PA-PSP onTAG biosynthesis may be cell-specific.

As discussed earlier, presqualene diphosphate is an intermediate insqualene formation and a precursor of cholesterol. Fukunaga et al [48]have shown that PA-PSP can dephosphorylate this precursor ofcholesterol in-vitro, and our results show that it increases TAGbiosynthesis. Taken together with the differential distribution of PA-PSP in several cells, it is tempting to speculate that overexpression ofPA-PSP in-vivo may impact the biosynthetic rate of cholesterol anddrive metabolic pathways towards TAG and away from cholesterol. Ithas been previously shown that in non-neural cells, deprivation ofcholesterol triggers the activation of sterol regulatory element-binding proteins (SREBPs), that bind on sterol regulatory elements(SREs) in the enhancers of genes encoding enzymes of cholesterolbiosynthesis, triglyceride biosynthesis, and lipid uptake, resulting inlipogenesis and TAG accumulation [61]. Therefore the triacylglyceroland cholesterol biosynthetic pathways may be interconnected not onthe biochemical pathway level, but through transcriptional regulationas well.

In summary, PA-PSP overexpression led to increased levels of PCand TAG, and decreased levels of PI, while PA-PSP and CCTα acted insynchrony to regulate cellular lipid metabolism. Thus the effects thatwe observed are due to the PA phosphatase activity of PA-PSP whichincreases the endogenous pool of DAG available to phospholipid andtriacylglycerol biosynthesis. Phospholipid and neutral lipid biosyn-

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thesis is a cellular process that responds to multiple intracellular andenvironmental stimuli. The conversion of PA to DAG is a keybranching point in the distribution of the glycerol backbone intolipid species. The evolution of multiple enzyme classes and genes thatwill regulate lipid formation by integrating and responding to diversesignals is manifested in the capacity of PA-PSP to modulatephospholipid and TAG biosynthesis. We propose that in addition tothe recently characterized type I PAP [11], there is a eukaryotic type IIPAP enzyme, PA-PSP, that participates in de-novo biosynthesis ofphospholipids and triacylglycerols.

Acknowledgments

This work was supported in part by a Marie Curie ReintegrationGrant to A.L, contract grant number 013030; G.L. has a BiomedicalResearch Foundation postdoctoral fellowship. We would like to thankKostas Zorpas for the technical assistance.

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