Gene Cloning and Characterization of CDP-diacylglycerol Synthasefrom Rat Brain*

(Received for publication, October 1, 1996, and in revised form, January 14, 1997)

Sachiko Saito‡§, Kaoru Goto‡, Akira Tonosaki§, and Hisatake Kondo‡¶

From the ‡Department of Anatomy, Tohoku University School of Medicine, Sendai 980-77 andthe §Department of Anatomy, Yamagata University School of Medicine, Yamagata 990-23, Japan

A cDNA encoded a 462-amino acid protein, whichshowed CDP-diacylglycerol synthase (CDS) activity wascloned for the first time as the vertebrate enzyme mol-ecule from rat brain cDNA library. The deduced molec-ular mass of this rat CDS was 53 kDa, and putativeprimary structure included several possible membrane-spanning regions. At the amino acid sequence level, ratCDS shared 55.5%, 31.7%, and 20.9% identity with al-ready known Drosophila, Saccharomyces cerevisiae, andEscherichia coli CDS, respectively. This rat CDS pre-ferred 1-stearoyl-2-arachidonoyl phosphatidic acid as asubstrate, and its activity was strongly inhibited byphosphatidylglycerol 4,5-bisphosphate. By immunoblot-ting analysis of COS cells overexpressed with theepitope-tagged for rat CDS, a 60-kDa band was detected.By epitope-tag immunocytochemistry, the CDS proteinwas mainly localized in close association with the mem-brane of the endoplasmic reticulum of the transfectedcells. The intense mRNA expression of CDS was local-ized in the cerebellar Purkinje cells, the pineal body,and the inner segment of photoreceptor cells. Addition-ally, very intense expression was detected in postmitoticspermatocytes and spermatids.

Phosphoinositide cycle mediates one of the intracellular sig-nal transduction pathways in eukaryotic cells and produces aclass of second messengers that are involved in cell growth (1,2), differentiation (3, 4), the action of hormones and neuro-transmitters (5), and sensory perception (6–8). Triggering ofthe cell surface receptors, such as G-protein-coupled receptorsand tyrosine kinase receptors, initiates the cycle by activatingphospholipase C (PLC),1 resulting in the hydrolysis of phos-

phatidylinositol 4,5-bisphosphate (PIP2) into two second mes-sengers, diacylglycerol (DAG) and inositol 1,4,5-triphosphate(IP3). DAG is subsequently phosphorylated by DAG kinase tosynthesize phosphatidic acid (PA), a presumed novel messen-ger (9–11). In this cycle, CDP-diacylglycerol synthase (CDS)(CTP-phosphatidate cytidylyltransferase) catalyzes the con-verting process from PA to CDP-DAG, the precursor to phos-phatidylinositol (PI), which is phosphorylated to synthesizePIP2 eventually.

The importance of CDS in the signal transduction has beenstrengthened in a recent study by Wu et al. (12), showing thatoverexpression of a photoreceptor cell-specific isoform of CDSin Drosophila increases the amplitude of the light response. Itsmutants (cds mutants) cannot sustain a light-activated currentand undergo light-dependent retinal degeneration, which canbe suppressed by a mutation in PLC. The difference in photo-transduction mechanism between vertebrate and invertebrateis well known, such as the primary second messenger role ofcGMP in vertebrate photoreceptors versus that of IP3 in inver-tebrate. However, recent investigations have shown uniquedistribution of phosphoinositide-specific PLC (13–15), proteinkinase C (16, 17), and IP3 receptor (18) in vertebrate rod outersegment, and that light may enhance the activity of PLC (19–22) and protein kinase C (23), and phosphoinositide synthesis(24). In addition, it has been reported that cytoplasmic Ca21

concentration mediates light adaptation in vertebrate photore-ceptors (25, 26). Thus it is suggested that in vertebrates as wellas invertebrates the phosphoinositide cycle may play a role inthe phototransduction signaling. It is therefore possible thatCDS is also important in the signal transduction mechanism ofvertebrate retina and neural cells.

As a step toward understanding the possible functional sig-nificance of this enzyme in vertebrate cellular signaling, thepresent study was attempted to perform molecular cloning of aCDS molecule from rat brain and to clarify its enzymatic fea-ture and tissue and cell localization. Our result shows that anewly identified CDS prefers arachidonate-containing PA as asubstrate, suggesting strongly a role for CDS in the phospho-inositide synthesis. It is also shown that the activity of CDS isinhibited by PIP2, suggesting that polyphosphoinositides reg-ulate their own synthesis through CDS activity. In addition, weshow the detailed localization at mRNA level in the retina andbrain, but unexpectedly the highest expression of its mRNA isdetected in the testis.

EXPERIMENTAL PROCEDURES

Polymerase Chain Reaction (PCR)—Total RNA was extracted fromadult Wistar rat brain by acid guanidinium thiocyanate-phenol-chloro-form extraction (27). Poly(A)1 RNA was isolated by chromatography onan oligo(dT)-cellulose column. First-strand cDNA was prepared usingFirst-Strand cDNA Synthesis kit (Pharmacia Biotech Inc.).

Primers used for PCR were composed of two degenerate oligonucleo-tides, which were made according to the amino acid sequences of con-served regions between Drosophila CDS (12) and E. coli CDS (28): the

* This study was supported by Grants-in-aid for Scientific Research07457001, 07278203, and 08270205 (to H. K.) and 07780670 and08780718 (to K. G.) from the Ministry of Education, Science and Cul-ture of Japan and by a grant from Nishijima Neurosurgery HospitalFoundation, Numazu, Japan. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submittedto the GenBankTM/EBI Data Bank with accession number(s)AB000517.

¶ To whom correspondence should be addressed: Dept. of Anatomy,Tohoku University School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sen-dai 980-77, Japan. Tel.: 81-22-717-8033; Fax: 81-22-717-8021.

1 The abbreviations used are: PLC, phospholipase C; PIP, phosphati-dylinositol 4-monophosphate; PIP2, phosphatidylinositol 4,5-bisphos-phate; PI, phosphatidylinositol; DAG, diacylglycerol; IP3, inositol 1,4,5-triphosphate; PA, phosphatidic acid; CDS, CDP-diacylglycerolsynthase; aa, amino acid(s); PCR, polymerase chain reaction; kb, kilo-base(s); di-C12:0-PA, 1,2-dilauroyl-sn-glycero-3-phosphate; di-C18:0-PA, 1,2-distearoyl-sn-glycero-3-phosphate; di-C18:1-PA, 1,2-dioleoyl-sn-glycero-3-phosphate; C18:0/C20:4-PA, 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphate.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 14, Issue of April 4, pp. 9503–9509, 1997© 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www-jbc.stanford.edu/jbc/ 9503

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regions corresponded to the amino acid sequences TW(E/Q)GFIGG (aa276–283 for 59 primer) and IPGHGGI(M/L) (aa 391–398 for 39 primer)(amino acid numbers represent those of Drosophila CDS). The se-quences for the primers were designed according to the mammaliancodon usage: GGAATTCAC(C/A)TGG(G/C)A(G/A)GG(C/A)TT(C/T)AT-(C/T)GG(C/A/G)GG for 59 primer and CGGGATCCA(T/G)(G/A)AT(G/T)CC(G/T)CC(G/A)TG(G/T)CC(G/A)GG(G/A)AT for 39 primer. The 59ends of 59 and 39 primers were designed to contain an EcoRI site and aBamHI site, respectively, for subsequent cleavage of subcloned cDNAfragments. PCR amplification was performed by using the first-strandcDNA and Ampli-Taq DNA polymerase according to following schedule:94 °C for 30 s, 55 °C for 45 s, and 72 °C for 2 min, for 30 cycles, followedby further incubation at 72 °C for 7 min. Subcloned cDNA fragmentswere sequenced by the model 373 DNA autosequencer (AppliedBiosystems).

cDNA Cloning—A cDNA library of adult rat brain on postnatal day49 was constructed as described previously (29). Clones (4 3 106)derived from the cDNA library were screened by hybridization in thepresence of 50% formamide at 42 °C with the subcloned PCR fragmentslabeled with [32P]dCTP. Washing conditions were carried out in 0.1 3SSC (SSC: 0.15 M NaCl, 0.015 M trisodium citrate), 0.1% sodium dodecylsulfate (SDS) at 42 °C. Among five clones isolated, one clone (pCDS4)containing the longest cDNA insert (3.5 kb) was selected for furthersequence analysis on both strands as described above.

Transfection and CDS Assay—A full-length cDNA (pCDS4) for thenewly cloned molecule was subcloned into the expression vector, pSRE(pcDL-SRa296, Refs. 30 and 31). The constructs or vector alone as acontrol was transfected into COS-7 cells by DEAE-dextran method (32).After incubation for 3 days in Dulbecco’s modified Eagle’s mediumsupplemented with 10% fetal calf serum, transfected cells were har-vested and lysed by sonication in lysis buffer (31). After removal ofundisrupted cells by centrifugation (550 3 g, 10 min, at 4 °C), resultingsupernatant was termed a total lysate. Protein concentrations weredetermined by the method of Lowry et al. (33), with bovine serumalbumin as a standard. CDS activity was measured by the mixed-micelle assay methods of Wu et al. (12) and Sparrow and Raetz (34) withsome modifications. The reaction mixture (50 ml) contained 0.1 M Tris-HCl (pH 7.5), 0.2 M KCl, 1 mg/ml bovine serum albumin, 5 mM (0.3%)Triton X-100, 0.25 mM dithiothreitol, 10 mM MgCl2, 0–1000 mM PA, 1mM [a-32P]CTP (105 cpm/nmol). In some experiments, [a-32P]dCTP wasused instead of [a-32P]CTP. PAs used as substrates for CDS are com-posed of PA from egg yolk lecithin: 1,2-dilauroyl-sn-glycero-3-phosphate(di-C12:0-PA) as a short chain PA, and 1,2-distearoyl-sn-glycero-3-phos-phate (di-C18:0-PA), 1,2-dioleoyl-sn-glycero-3-phosphate (di-C18:1-PA),and 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphate (C18:0/C20:4-PA) as long chain PAs. To analyze the sensitivity of CDS activity tophosphoinositide, PI, PIP (phosphatidylinositol 4-monophosphate), andPIP2 (Sigma) were added to the reaction mixture at concentration of 2,5, 10, and 20 mol%. The reaction was continued for 10 min at 30 °C andwas stopped with 100 ml of 1 N HCl. A two-phase system was created byadding 250 ml of chloroform/methanol (1:1, v/v), and the mixtures werethoroughly vortexed and then centrifuged. The lower organic phase waswashed with 100 ml of chloroform, 1 N HCl (1:1, v/v) and was analyzedby thin layer chromatography which was developed in a solution con-taining chloroform/methanol/water/acetic acid (50:28:8:4, v/v/v/v). 1,2-Diacyl-sn-glycero-3-diphosphocytidine (CDP-DAG) from egg yolk leci-thin (Sigma) was used as a marker. The bands of CDP-DAG detected byautoradiography was scraped with a sharp spatula and collected forliquid scintillation counting.

Immunoblotting and Immunocytochemistry by Epitope-tagging—Anepitope-tag composed of eight amino acids (FLAG marker peptide,Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys; Eastman Kodak Corp.) was fusedto the newly identified molecule by cloning the 24 base pairs of FLAGcoding sequence up- or downstream to the coding region of the novelcDNA. The FLAG epitope-tagged molecules were expressed in COS-7cells using the expression vector, pSRE, by the DEAE-dextran method(30–32). Total lysate of the overexpressed COS-7 cells was prepared asdescribed above. Total lysate boiled for 5 min in Laemmli’s samplebuffer was subjected to 10% SDS-polyacrylamide gel electrophoresis(35). The separated proteins were then electrophoretically transferredto a nitrocellulose membrane (0.45-mm pore size). After blocking thenonspecific binding sites in the 5% skim milk (w/v) in phosphate-buffered saline, 0.01% Tween 20, the membrane was incubated for 1 hat room temperature with antibody against FLAG and then treatedwith peroxidase-conjugated anti-mouse IgG antibody for 1 h. The im-munoreactive bands were detected using a chemiluminescence detec-tion kit (ECL Western blotting detection kit, Amersham).

Transfected cells were fixed with 4% paraformaldehyde, 0.1 M so-

dium phosphate buffer (pH 7.2), 0.2% Triton X-100, or fixed with thesame fixative without the detergent and freeze-thawed after immersionin 40% sucrose. The cells were incubated with the anti-FLAG antibody(Kodak) and sites of the antigen-antibody reaction were visualizedusing the avidin-biotinylated peroxidase complex (ABC) system (VectorLaboratories) with 3,39-diaminobenzidine tetrahydrochloride as a sub-strate. Some of the freeze-thawed specimens, after immunoreaction,were postfixed with 1% OsO4, treated with 0.5% uranyl acetate, andembedded in Epon 812. Ultrathin sections were examined under elec-tron microscope.

Northern Blot Analysis—Total RNAs were extracted from adult ratbrains, eyeballs, and several other tissues as described above. Each of thetotal RNA samples (30 mg/lane) was denatured with formamide andsize-separated by formaline/agarose gel electrophoresis. The RNAs weretransferred and fixed to nylon membranes (Nytran, Schleicher & Schuell)and hybridized with a probe corresponding to the 39 non-coding sequences(nucleotides 1423–1875) labeled with [32P]dCTP. Conditions for hybrid-ization and washing were performed as described previously (29).

In Situ Hybridization Histochemistry—Fresh frozen blocks of adultrat brains, eyeballs, and testes were sectioned at 10 or 25 mm thicknesson a cryostat. The sections were mounted on silane-coated glass slides,fixed with 4% paraformaldehyde, 0.1 M sodium phosphate buffer (pH7.2), pretreated and hybridized as described previously (29). Two kindsof probes were used in the experiment: a cDNA probe corresponding tonucleotides 526–1311 labeled with [a-35S]dATP by nick translation anda probe composed of 45-mer antisense oligonucleotides complementaryto a part of 39 non-coding sequences (nucleotides 1423–1467) labeledwith [a-35S]dATP by terminal deoxynucleotidyl transferase. After hy-bridization at 42 °C for 18 h, the slides were sequentially rinsed twice in2 3 SSC, 0.1% Sarkosyl at 42 °C for 15 min each, three times in 0.1 3SSC, 0.1% Sarkosyl at 42 °C for 40 min each, and dehydrated in 70%and 100% ethanol containing 0.3 M ammonium acetate. The sectionswere autoradiographed using NTB2 nuclear track emulsion (Kodak) for15–60 days.

RESULTS

The nucleotide and deduced amino acid sequences of thecomposite CDS cDNA are presented in Fig. 1. The putativeinitiation codon was preceded by in-frame stop codons. Thepredicted open reading frame encoded a protein of 462 aminoacids with the deduced molecular mass of 53 kDa. The deducedamino acid sequence of this rat CDS shared 55.5%, 31.7% and20.9% identity to that of Drosophila (12), Saccharomyces cer-evisiae (36), and Escherichia coli (28), respectively (Fig. 2a).Near the carboxyl terminus, domains sharing more than 80%identity to each other were contained in all molecules. Severalprotein kinase-recognition sequence motifs were identifiedsuch as those recognized by myosin-I heavy chain kinase (aa345–348), multifunctional Ca21/calmodulin-dependent proteinkinase II (aa 52–57), cGMP-dependent protein kinase (aa 312–318), proline-dependent protein kinase (aa 265–268 and 271–274), casein kinase I (aa 76–80), and glycogen synthase kinase3 (aa 312–318) (37). Hydrophobicity analysis by the method ofKyte and Doolittle (38) revealed that this novel molecule, likethe other three CDSs, was very hydrophobic and appeared tocontain several possible membrane-spanning regions, asjudged by the fact that at least 19 sequential residues have anaverage hydrophilic value of greater than 11.6 (Fig. 2b). Theamino terminus of this molecule was hydrophilic, similar tothose of CDSs of Drosophila and S. cerevisiae.

By measurement of the CDS activity of this novel molecule,the total lysate from COS-7 cells transfected with the fulllength of the present cDNA showed about 5–7 times higheractivity toward PAs than the control total lysate (e.g. 184 6 9.3;28 6 1.5 pmol/min/mg total protein under the assay conditionat 200 mM C18:0/C20:4-PA). The enzymatic activity of thismolecule was the highest toward 1-stearoyl-2-arachidonoyl-PA(C18:0/20:4-PA) among the others, whereas little or no activitywas detected toward PAs containing saturated fatty acylgroups in both of the sn-1 and -2 positions (di-C12:0-PA, di-C18:0-PA) (Fig. 3). From double-reciprocal (Lineweaver-Burk)plots, values for the apparent Km toward C18:0/C20:4-PA, di-

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C18:1-PA, and PA from egg yolk lecithin were 102, 114, and 138mM, respectively; and for the apparent Vmax were 268, 259, and198 pmol/min/mg total protein, respectively. Values for appar-ent Vmax/Km (specificity constant) calculated from Eadie-Hof-stee plots were 2.51, 2.28, and 1.48, respectively. When theutilization of CTP and dCTP for the activation of phospholipidintermediates was compared, dCTP was incorporated intophospholipid precursors at almost the same rate as CTP. In theanalysis of inhibitory effects of PI, PIP, and PIP2 on the CDSactivity of the present molecule, the activity decreased by 50%at 2 mol% PIP2 and by 80% at 10 mol% and more (Fig. 4), underthe assay condition at 1000 mM C18:0/C20:4-PA (the present

molecule showed the maximum activity at 200–1000 mM C18:0/C20:4-PA). In contrast, the activity decreased by at most 40%at 5–20 mol% PIP. Addition of PI at various concentrationsfrom 2 to 20 mol% produced little effects on CDS activity.Similar results were obtained in the experiments for C18:0/C20:4-PA and di-C18:1-PA at 100 or 500 mM (data not shown).

In the immunoblotting of epitope (FLAG)-tagged CDS withthe antibody against the FLAG tag, a single immunoreactionband was observed at size of 60 kDa (Fig. 5).

In the immunocytochemistry (Fig. 6a) of the transfectedCOS-7 cells with the antibody against FLAG tag, immunore-active cells accounted for approximately 20–40% of the total

FIG. 1. Nucleotide sequence of the composite cDNA and the deduced primary structure of rat CDS. In-frame stop codons in 59non-coding region are underlined.

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cell population and they were randomly dispersed in each cul-ture dish. The immnoreactivity was localized widely through-out the cells in forms of delicate networks composed of fine dotswith a tendency of condensation in one pole of juxtanuclearcytoplasm, and they were also deposited along the nuclear rim.In immunoelectron microscopy (Fig. 6b), the immunoreactivitywas detected along the membranes of endoplasmic reticulum,vesicles and vacuoles, and nuclear envelopes. No immunoreac-tivity was detected in any cells when the transfection was madeof the cDNA without the FLAG tag.

In Northern blot analysis (Fig. 7) of adult rat on postnatal

day 49, the hybridization bands were detected very strongly intestis at sizes of 3.5 and 2.2 kb, suggesting a possibility ofalternative splicing. The hybridization band was strongly de-tected in eyeball and brain at a size of 3.5 kb. Among othertissues, a weak band of 3.5 kb was detected in kidney, smallintestine, and placenta, whereas a faint band of 3.5 kb was seenin thymus and lung after long exposure. Any hybridizationbands could not be detected in heart, liver, and ovary.

By in situ hybridization histochemical analysis of the adultbrain (Fig. 8), the most intense expression signals were detectedin the cerebellar Purkinje cells and intense expression was found

FIG. 2. a, identity of the amino acid se-quence among rat, Drosophila, S. cerevi-siae, and E. coli CDSs. Conserved resi-dues are stippled. b, linear representationof molecular structure of the CDSs de-scribed above. Hydrophilic regions, andpossible membrane-spanning regions areshown.

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in the pineal body. Moderate to low expression was seen in layersII–VI of the cerebral cortex, in the hippocampal pyramidal celllayer and subiculum, and the olfactory mitral cells. Low expres-sion was seen in almost all neurons in the fore-, mid- and hind-brains with much lower expression in the caudate putamen. Lowexpression was also seen in the choroid plexuses. No significantexpression was detected in the white matter including the corpuscallosum and cerebellar medulla or in the glia limitans. In theretina, positive expression signals were confined to the innersegment of the photoreceptor cells (Fig. 9).

In the adult testis, the hybridization signals were denselydeposited in most of the adluminal compartment of the seminif-erous tubule composed of postmitotic spermatocytes and sperma-tids, while no significant signals were detected in the apicalcompartment composed of spermatozoa or in the basal compart-ment composed of proliferating spermatogonia. No significantsignals were found in the interstitial cell clusters (Fig. 10).

In the control experiment in which several random sectionsof brain, retina, and testis were hybridized with the labeledoligonucleotide probe in the presence of 100-fold excessamounts of the unlabeled probe, no significant expression sig-nals were detected in any regions of the tissue sections.

DISCUSSION

We report here for the first time the molecular cloning ofvertebrate CDS and clarify its primary structure, enzymaticfeatures, and localization in detail. The primary structure ofthis rat CDS shared 55.5%, 31.7%, and 20.9% identity to that ofDrosophila (12), S. cerevisiae (36), and E. coli (28), respectively(Fig. 2a). The high hydrophobicity and the inclusion of severalputative membrane-spanning regions in the primary structureof the present molecule and the localization associated with the

endoplasmic reticulum and nuclear envelopes are well consist-ent with available biochemical data of the mammalian CDS sofar reported (34, 39–41). The deduced molecular size of 53 kDais smaller than the size of 60 kDa detected by epitope-taggedimmunoblotting. This discrepancy may be ascribed to the post-translational modification of CDS in vivo. From the sequence,it is clear that there are several potential serine/threoninephosphorylation sites, suggesting the possibility that phospho-rylation by serine/threonine kinases may be involved in regu-lation of this enzyme activity.

The present study shows the substrate specificity of rat CDStoward 1-stearoyl-2-arachidonoyl PA (C18:0/C20:4-PA) amongseveral PAs (Fig. 3). In mammalian cells, CDP-DAG is used toproduce phosphatidylinositol, phosphatidylglycerol, and cardi-olipin, and CDP-DAG is therefore an important regulatorybranching point in the phospholipid metabolism. Since the PIpredominantly consist of the 1-stearoyl-2-arachidonoyl species

FIG. 3. CDS activity toward various PAs in total lysates ofCOS-7 cells transfected with pSRE-rat CDS cDNA. The insetshows double-reciprocal (Lineweaver-Burk) plots of PA with differentfatty acyl composition dependence. Values for the apparent Km towardC18:0/C20:4-PA, di-C18:1-PA, and PA from egg yolk lecithin were 102,114, and 138 mM, respectively; and for the apparent Vmax were 268, 259,and 198 pmol/min/mg total protein, respectively. (Values for apparentVmax/Km (specificity constant) calculated from Eadie-Hofstee plots were2.51, 2.28, and 1.48, respectively.) The CDS activity for control lysate is28 6 1.5 pmol/min/mg total protein toward C18:0/C20:4-PA under theassay condition at 200 mM C18:0/C20:4-PA. Values shown are means 6S.D. (n 5 6).

FIG. 4. Effects of PIP2 (●), PIP (E), and PI (3) on rat CDSactivity under the assay condition at 1000 mM C18:0/C20:4-PA.Each point represents the average of three determinations performed induplicate. The error ranges of all data are within 10%. Similar resultwere obtained in the experiments for C18:0/C20:4-PA and di-C18:1-PAat 100 or 500 mM (data not shown).

FIG. 5. Immunoblot of total lysate extracted from the cellsoverexpressed with pSRE vector only (lane 1), and epitope-tagged rat CDS cDNA (lane 2). Immunoreaction was performedusing anti FLAG-tag antibody.

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(42), the present substrate specificity of rat CDS suggestsstrongly that this enzyme molecule selectively participates inthe phosphoinositide cycle, but not in the synthesis of phos-phatidylglycerol and cardiolipin.

The present study also clarifies the marked inhibition of ratCDS activity in vitro by PIP2 (Fig. 4), a presumed end productof the phosphoinositide cycle. This represents another newexample of the potential for acidic phospholipids includingphosphoinositides to modulate the in vitro activities of severalmembrane enzymes related to the signal transduction. PI-4-phosphate 5-kinase appears to be inhibited by PIP2 and beactivated by PA (43–45), casein kinase I appears to be inhibitedby PIP2 (46), phosphoinositide-specific PLC-g1 appears to beactivated by PA (47), and several protein kinase C isoform areactivated by PIP2 (phosphatidylinositol 3,4-bisphosphate aswell as phosphatidylinositol 4,5-bisphosphate) and phosphati-dylinositol 3,4,5-trisphosphate (48–50). This PIP2-induced en-zymatic inhibition and the substrate specificity of rat CDSsuggest the presence of a mechanism for PIP2 to regulate itsown synthesis through the phosphoinositide cycle by feedback.

The PIP2-induced inhibition has recently been demonstratedto occur on arachidonoyl-DAG kinase, which catalyzes the syn-thesis of PA from sn-1-acyl-2-arachidonoyl-DAG selectively byphosphorylation (51). Considering the fact that DAG kinaseand CDS are sequentially involved in the phosphoinositide

resynthesis, the finding that both arachidonoyl-specific DAGand CDS are inhibited by PIP2 strongly suggests that PIP2 issynthesized on demand as opposed to being stored, and theconversion process from DAG to CDP-DAG through PA, theinitial steps of resynthesis of phosphoinositides, is tightly reg-ulated in the phosphoinositide-mediated signaling cascades. Inaddition, similar to the CDS molecule, the arachidonoyl-DAGkinase has been shown to be expressed also highly in the testisas well as the brain and to be an integral membrane protein(52). It is thus suggested that these two arachidonoyl-specificenzyme molecules are located in the same subcellular mem-branes and are regulated by the lipid microenvironment withinthe membranes in a paired fashion in the two consecutivereactions of the phosphoinositide cycle. The detailed cellular

FIG. 6. Immunocytochemistry of the COS-7 cell transfectedwith epitope-tagged rat CDS cDNA using anti FLAG-tag anti-body. a, light micrograph, bar 5 10 mm. b, electron micrograph (orig-inal magnification, 35000), bar 5 500 nm. Arrows show typical immu-nodeposits. N, nucleus; M, mitochondria; PM, plasma membrane.

FIG. 7. Northern blot analysis of rat CDS mRNA in various rattissues on postnatal day 49. Size markers (arrowhead) represent 28and 18 S rRNAs.

FIG. 8. In situ hybridization of rat CDS mRNA in adult ratbrain. a, dark field micrograph of parasagittal section through caudateputamen (CP). OB, olfactory bulb; Co, cerebral cortex; H, hippocampalformation; Th, thalamus; SC, superior colliculus; IC, inferior colliculus;Pn, pontine nuclei; Cb, cerebellar cortex (bar 5 2 mm). b, dark fieldmicrograph of sagittal section through the pineal body (P) (bar 5 1 mm).c, bright field micrograph of higher magnification of cerebellar cortex.Mo, molecular layer; Pk, Purkinje cell; Gr, granular layer. (Hematoxy-lin stain, 25 mm thickness, autoradiographed for 60 days; bar 5 50mm.).

FIG. 9. Phase-contrast micrograph (a) and dark field light mi-crograph (b) of rat CDS mRNA expression in the adult retina byin situ hybridization. GC, ganglion cell layer; IP, inner plexiformlayer; IN, inner nuclear layer; OP, outer plexiform layer; ON, outernuclear layer; IS, inner segment; OS, outer segment; PE, pigmentepithelium. Note significant expression for CDS mRNA in the innersegment. (Hematoxylin stain, 25 mm thickness, autoradiographed for60 days; bars 5 50 mm.).

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and subcellular localization of the arachidonoyl-DAG kinase inthe testis as well as the brain and retina, and its comparisonwith that of this CDS may be informative in this regard. Thefunctional significance of the most intensive expression of ratCDS mRNA in testis, especially in postmitotic spermatocytesand spermatids remains to be elucidated.

The localization of this CDS mRNA in the inner segment ofrat photoreceptor cells (Fig. 9) corresponds well to the presenceof the Drosophila homologue in the photoreceptor cells, sug-gesting some similar functional significance between the twohomologous molecules of far remote animal species. As cited inthe Introduction, it has clearly been demonstrated that phos-phoinositide-specific PLC isoforms cloned from mammalianretina are distributed in the photoreceptor cells (13–15) andthat light induces the decrease in PIP2 and increase in IP3 inthe photoreceptor cells (19–22), and light also enhanced syn-thesis of PI by activation of phosphoinositide cycle in these cells(24). The existence of IP3 receptor in rod outer segment has alsobeen shown (18), and Ca21-mediated light adaptation has beendemonstrated in vertebrate photoreceptor cells (25, 26). Inaddition, protein kinase C, an enzyme activated by DAG, isreportedly present in the rod outer segment (16, 17), and itsactivity appears to be stimulated by light (23). Protein kinase Cis known to phosphorylate several rod outer segment proteins,including rhodopsin (53) and phosphodiesterase g (54). There-fore, despite the known difference in the primary second mes-senger species, cGMP versus IP3, between vertebrate andinvertebrate phototransduction, our finding implies the pos-sibility that some additional roles for the phototransduction areplayed by this CDS molecule through the phosphoinositidecycle in rat photoreceptor cells. The intense gene expression ofthis CDS in the pineal organ (Fig. 8b), a phylogenically homol-ogous organ to the retina, is in favor of this possibility. Theheterogeneous but wide expression localization of CDS mRNAin the gray matter of the brain (Fig. 8a) is also instructive forthe functional significance of CDS in the neuronal signal trans-duction. The intense expression of CDS mRNA in the cerebellarPurkinje cells and moderate expression in the hippocampalpyramidal cells are consistent with the gene expression patternof the receptor for IP3, one of the two second messengers pro-duced by phosphoinositide cycle (55).

The molecular identification of the first mammalian CDScompleted by the present study would accelerate further eluci-dation of the functional significance of this enzyme in thephosphoinositide-related signal pathway.

Acknowledgment—We thank Dr. Hiroshi Munakata (Department of

Biochemistry, Tohoku University School of Medicine) for helpful adviceand suggestions for this work.

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FIG. 10. Bright field light micrograph (a) and dark field lightmicrograph (b) of rat CDS mRNA expression in the seminiferoustubule of adult rat testis by in situ hybridization. Note intensehybridization signals in the zone of postmitotic spermatocytes (pSc) andspermatids (St). Sg, spermatogonia; Sz, spermatozoa; I, interstitium.(Hematoxylin stain, 25 mm thickness, autoradiographed for 30 days;bars 5 200 mm.).

CDP-diacylglycerol Synthase 9509

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Sachiko Saito, Kaoru Goto, Akira Tonosaki and Hisatake KondoGene Cloning and Characterization of CDP-diacylglycerol Synthase from Rat Brain

doi: 10.1074/jbc.272.14.95031997, 272:9503-9509.J. Biol. Chem. 

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