J Biol Chem, Vol. 274, Issue 38, 26992-27001, September 17, 1999

Distribution of CTP:Phosphocholine Cytidylyltransferase (CCT) Isoforms
IDENTIFICATION OF A NEW CCT SPLICE VARIANT^*

Athanasios Lykidis, Irina Baburina^§, and Suzanne Jackowski^¶

From the  Department of Biochemistry, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 and the ^¶ Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CTP:phosphocholine cytidylyltransferase is a major regulator of phosphatidylcholine biosynthesis. A single isoform, CCT, has been studied extensively and a second isoform, CCT, was recently identified. We identify and characterize a third cDNA, CCT2, that differs from CCT1 at the carboxyl-terminal end and is predicted to arise as a splice variant of the CCT gene. Like CCT, CCT2 is heavily phosphorylated in vivo, in contrast to CCT1. CCT1 and CCT2 mRNAs were differentially expressed by the human tissues examined, whereas CCT was more uniformly represented. Using isoform-specific antibodies, both CCT1 and CCT2 localized to the endoplasmic reticulum of cells, in contrast to CCT which resided in the nucleus in addition to associating with the endoplasmic reticulum. CCT2 protein has enzymatic activity in vitro and was able to complement the temperature-sensitive cytidylyltransferase defect in CHO58 cells, just as CCT and CCT1 supporting proliferation at the nonpermissive conditions. Overexpression experiments did not reveal discrete physiological functions for the three isoforms that catalyze the same biochemical reaction; however, the differential cellular localization and tissue-specific distribution suggest that CCT1 and CCT2 may play a role that is distinct from ubiquitously expressed CCT.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

PtdCho¹ is the major membrane phospholipid in higher eukaryotes and is also secreted by particular tissues for important extracellular tasks. For example, it is a significant component of lung surfactant, serum lipoproteins, and bile. CCT is a key regulator of PtdCho biosynthesis (1) and membrane-protein interaction is one important mechanism that governs cellular CCT activity (1, 2). Recently a second isoform, CCT, was discovered which is encoded by a second gene (3). CCT and CCT have nearly identical amino acid sequences in the catalytic domain which extends approximately from residues 72 to 233 in both proteins, and also near identity in the membrane-interaction domain which extends approximately from residues 256 to 288. Both isoforms are dependent on interaction with phospholipids for catalytic activity (3-9), as would be predicted from the high degree of identity in the membrane-interaction domains. These domains are characterized by three 11-residue amphipathic repeats that form -helices upon association with phospholipid regulators (10-13).

The amino terminus of CCT bears no resemblance to the amino terminus of CCT and does not include a nuclear localization sequence as was identified in the CCT protein (14, 15). CCT has been localized predominantly in the nucleus but the physiological significance of the nuclear localization of CCT remains unclear. CCT protein was localized outside the cell nucleus by indirect immunofluorescent microscopy (3). CCT consists of 330 amino acids, in contrast with the 367 residues of CCT, and lacks most of the carboxyl-terminal phosphorylation domain that is found in the CCT protein (9, 16). Phosphorylation of CCT interferes with the lipid stimulation of enzyme activity in vitro (17) and correlates with a reduction of PtdCho biosynthesis in vivo (18-24). Despite the differences at the amino and carboxyl termini of the proteins, both CCT and CCT exhibit high activity when overexpressed in COS-7 cells (3, 9, 25, 26) resulting in accumulation of cellular CDP-choline and increased radiolabeling of PtdCho (3, 27).

In this work we identify a third isoform of CCT, called CCT2, which is a splice variant of CCT. CCT2 encodes a 369-amino acid protein which is identical to the CCT1 isoform described previously from amino acids 1 to 320. However, CCT2 also has a carboxyl-terminal sequence that resembles the phosphorylation domain of CCT. The existence of two distinct CCT genes and two CCT splice variants raises the possibility of regulation of CCT activity at the level of gene expression as well as subcellular localization and phosphorylation (3). Thus, we investigated the expression of the CCT isoforms in human tissues, determine whether CCT2 has a phosphorylated carboxyl-terminal domain, and whether these structural differences alter the cellular localization of or the ability of CCT isoforms to complement defective CCT activity in vivo (28).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Sources of supplies were: Accurate Chemical & Scientific Corp., anti-mouse protein disulfide isomerase antibody; American Radiolabel Co., Inc., phospho-[methyl-¹⁴C]choline (specific activity, 55 mCi/mmol); Amersham Pharmacia Biotech, [³⁵S]methionine (specific activity, >1000 Ci/mmol); Life Technologies, Inc., LipofectAMINE reagent; Molecular Probes, Oregon Green^TM 488; Texas Red^TM, Hoechst and FluoReporter^TM labeling kits, Oregon Green^TM, and Texas Red^TM, concanavalin A, and wheat germ agglutinin conjugates, Prolong^TM antifade kit with mounting medium; Nalge Nunc International, LabTek^TM II Chamber Slides^TM; Promega, restriction endonucleases and other molecular biology reagents; Invitrogen, pcDNA3 vector plasmid, cDNA cycle kit, human poly(A)⁺ RNAs; Genome Systems, Inc., cDNA clone AA683266; Research Genetics, Inc., cDNA clone AI041180; Sigma, anti-FLAG M2 monoclonal antibody, CTP, phosphocholine and buffers; Analtech, thin-layer chromatography plates. All other supplies were reagent grade or better.

Antibodies-- Anti-CCT rabbit polyclonal antiserum was raised against a synthetic peptide (MDAQSSAKVNSRKRRKE) corresponding to the first 17 amino acids of CCT. Anti-CCT antibody (B1 epitope) was a rabbit polyclonal antiserum raised against a peptide (MEEIEHTCPQPRL) corresponding to amino acids 27-39 of CCT1 and CCT2. The anti-CCT antibody (B2 epitope) was a rabbit polyclonal antiserum raised against a synthetic peptide (TTDAESETGIPKSLSNEP) corresponding to amino acids 5-22 of CCT1 and CCT2. Anti-CCT2 antibody (B3 epitope) was a rabbit polyclonal antiserum raised against a synthetic peptide (PPSSPKAASRSISSMSEGD) corresponding to amino acids 347-365 of CCT2. Resequencing of the CCT2 clone identified that the correct residue at position 10 of the B3 peptide is an alanine instead of an arginine. The B1 and B2 epitope antibodies recognized both CCT1 and CCT2 whereas the B3 epitope antibody recognized only CCT2. Peptides and peptide antigens were prepared by the Molecular Resource Center of St. Jude Children's Research Hospital. The B1 and B2 antigens were prepared by coupling each peptide to keyhole limpet hemocyanin via an additional cysteine at the carboxyl terminus of the peptide whereas the B3 antigen was coupled at the amino terminus. Immunization of rabbits and collection of antiserum was performed by Rockland, Inc., according to their standard schedule. Antisera were purified by affinity chromatography on Affi-Gel 10 cross-linked to the peptide as described previously (3).

Isolation of the CCT2 cDNA and Construction of Expression Plasmids-- The EST data base was searched using the published CCT sequence (GenBank^TM/EBI Data Bank accession number AF052510). A clone from human brain was identified (GenBank^TM accession number AA683266) and purchased from Genome Systems. The cDNA sequence was determined on both strands using primers that flanked the multiple cloning sites and internal primers that were synthesized to ensure a complete read on both strands. A second EST clone from human testis was identified (GenBank^TM accession number AI041180) and purchased from Research Genetics. The cDNA sequence of the second clone was also determined. Clone AA683266 was subcloned into pcDNA3 using BamHI and XhoI (pAL1). pcDNA3 has an SspI site approximately 1 kilobase from the 5' end of the T7 promoter and pAL1 retains the SspI site of CCT. The cDNA encoding CCT1 in pcDNA3 (pPJ34) (3) was also digested with SspI. The approximately 1.1-kilobase fragment derived from plasmid pPJ34 was ligated to the 5.5-kilobase fragment of pAL1 to generate pAL2.

Construction of the CCT1(M27A) Mutant-- The M27A point mutation was constructed using overlap extension PCR with the CCT1 cDNA as template in pBlueScript SK and using the primers: M13 reverse: 5'-CAGGAAACAGCTATGACC-3', M27A forward: 5'-CAGAAACCGCGGAGGAAATAGAGC-3', M27A reverse: 5'-ATTTCCTCCGCGGTTTCTGAG-3', and SnaB1 reverse: 5'-AGGGAGCATCTCTGATAACTTCGTC-3'. Primers M27A forward and M27A reverse replace the ATG codon for methionine 27 with GCG encoding alanine. In the first round of PCR the pairs of primers M13rev-M27Arev and M27Afor-SnaB1rev generated products of 280 and 381 bp, respectively. 10 ng of these products were gel purified and used as template for the second round of PCR with primers M13rev and SnaB1rev. The 642-bp product was cloned into pCR2.1 (plasmid pPJ76) and sequenced to verify that it had the desired mutation. The BamHI-SnaBI fragment of pPJ76 was ligated into the CCT1 cDNA replacing the BamHI-SnaBI fragment of AA382871. The mutated CCT1 cDNA was subcloned into pcDNA3 using BamHI-XhoI.

CCT Assay-- CCT activity was determined essentially as described previously (3). The standard assay contained 150 mM bis-Tris-HCl, pH 6.5, 10 mM MgCl₂, 4 mM CTP, 64 µM lipid activator (PtdCho:oleic acid, 1:1), 1 mM phospho[¹⁴C]choline (specific activity 4.5 mCi/mmol) in a final volume of 50 µl. The reaction mixture was incubated at 37 °C for 10 min. The reaction was stopped by the addition of 5 µl of 0.5 M Na₃EDTA, and the tubes were vortexed and placed on ice. Next, 40 µl of each sample was spotted on preadsorbent Silica Gel G thin layer plates, which were developed in 2% ammonium hydroxide, 95% ethanol (1:1, v/v). CDP-[¹⁴C]choline was identified by comigration with a standard, scraped from the plate, and quantified by liquid scintillation counting. Protein was determined according to the Bradford method (29).

Transfection Experiments-- COS-7 cells were grown in 100-mm dishes to 80% confluency in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% glutamine. CHO58 cells were grown in 100-mm dishes at 33 °C in Ham's F-12 medium supplemented as above. Transfections using LipofectAMINE reagent were performed according to the manufacturer's instructions. Briefly, 10 µg of plasmid and 60 µl of LipofectAMINE reagent were diluted separately into 0.8 ml of serum-free medium. The two solutions were combined and incubated at room temperature for 45 min. Next, 6.4 ml of serum-free medium was added to each tube and the diluted solution was overlaid onto cells that had been previously rinsed with serum-free medium. The cells and reagents were incubated at 37 °C for 5 h, and then 8 ml of growth medium containing twice the normal amount of serum was added. The medium was replaced 24 h after the start of the transfection procedure. COS-7 cells were incubated for an additional 24 h at 37 °C and then harvested for analysis. CHO58 cells were transferred to 40 °C. After incubation for an additional 72 h at the restrictive temperature, CHO58 cells were washed twice with 10 ml of phosphate-buffered saline, cells were fixed by incubation for 5 min in CH₃OH/H₂O/CH₃COOH (45:45:10, v/v). After removal of the solvent, cells were incubated for 5 min in 0.05% Coomassie Blue R-250 in CH₃OH/H₂O/CH₃COOH (45:45:10 v/v) to stain colonies. Finally, dishes were washed twice with CH₃OH/H₂O/CH₃COOH (45:45:10, v/v) and pictures were taken.

Metabolic Labeling-- COS-7 cells were grown in 100-mm dishes and transfected with 10 µg of vector expressing CCT1, CCT2, or a pcDNA3 control vector without a cDNA insert. Cells were washed with PBS 48 h after transfection and fresh medium was added containing 1.6 mCi/dish of [³²P]orthophosphate. Cells were incubated for 60 min and then immunoprecipitated (see below).

Isolation of Poly(A)⁺ RNA from HeLa Cells-- HeLa cell cultures were harvested and total RNA was isolated by a guanidine isothiocyanate lysis procedure followed by pelleting RNA by CsCl gradient centrifugation (30). RNA pellets dissolved in 10 mM Tris-HCl, pH 7.5, 5% -mercaptoethanol, 0.5% Sarcosyl, 0.5% SDS, and 5 mM EDTA were extracted with phenol:chloroform:isoamyl alcohol (24:24:1, v/v) and precipitated with 2 volumes of ethanol. Poly(A)⁺ RNA was isolated by passing the total RNA through an oligo(dT) column (Amersham Pharmacia Biotech) as described by standard protocols (31).

RNA Analysis-- RT-PCR was performed using human poly(A)⁺ RNA that was purchased from Invitrogen, Inc., or using poly(A)⁺ RNA isolated from HeLa cells. The cDNA cycle kit (Invitrogen) was used to synthesize the first strand of cDNA following manufacturer's recommended procedure. Poly(A)⁺ RNA (1 µg) from each source was used in each 20 µl of reaction with random and oligo(dT) primers. The two tubes, where the reverse transcriptase reaction was performed, were combined and 5 µl of the first strand cDNA synthesis mixture was used for PCR amplification of CCT sequences. The forward primer for detection of CCT expression was 5'-GAAGGTGGAGGAAAAAAG-3' corresponding to 795-812 bp of the CCT cDNA sequence, and the reverse primer was 5'-ACAGAAAGGGAGGACAG-3' corresponding to 1123-1159 bp of the CCT cDNA sequence. The forward primer for CCT was 5'-CAAGTGGACAAAATGAAGG-3' corresponding to 733-751 bp of the CCT cDNA sequence and the reverse primer was 5'-CTAGAAGTCTCTGCACCTCG-3' corresponding to 1299-1238 bp of the CCT2 sequence or 974-993 bp of the CCT1 sequence. The PCR was performed in 50-µl reaction volume with 35 thermocycles at 94 °C for 1 min, 56 °C for 2 min, and 72 °C for 2 min. The PCR products were separated by agarose gel electrophoresis.

Transcription-translation Analysis-- Plasmid DNA was isolated, transcribed, translated, and labeled with [³⁵S]methionine using the Promega T7-coupled transcription/translation kit according to the manufacturer's instructions. The labeled proteins were analyzed by SDS-gel electrophoresis and visualized by autoradiography.

Immunoblots and Immunoprecipitation-- Cell lysates (50 µg of protein) were separated by SDS-gel electrophoresis on 12% polyacrylamide gels and transferred by electroblotting onto nitrocellulose membranes. Immunoblotting was performed by incubation of the membranes with purified anti-CCT (1:2000 dilution), purified anti-CCT1 (B2 epitope) (1:2000 dilution), or purified anti-CCT2 (B3 epitope) (1:2000 dilution) as primary antibody. The Amersham Pharmacia Biotech ECL Western blotting reagents and protocol were used to identify the immunoreactive proteins. For immunoprecipitations, cells were washed twice with PBS and lysed in the culture dish with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 2% aprotinin, 5 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 100 µM Na₃VO₄) for 30 min in 4 °C with gentle agitation. Cell lysates and debris were scraped from the dish and centrifuged for 10 min at 10,000 × g at 4 °C. Lysate supernatants were incubated for 1 h with 8 µg of anti-CCT (B2 epitope) purified antibody at 4 °C and then with the protein A-Sepharose beads pre-equilibrated in lysis buffer for 1 h at 4 °C. The beads were collected and washed thoroughly. Immune complexes were disrupted by addition of Laemmli buffer and heated in boiling water for 3 min. Proteins were separated by SDS-gel electrophoresis and phosphoproteins were detected by autoradiography.

Fluorescent Labeling of Affinity Purified Antibodies-- The antibodies were labeled according to the instructions provided with Molecular Probes' FluoReporter^TM labeling kits. Briefly, 200 µl of the 1-2 mg/ml antibody in PBS was combined with 20 ml of 1 M sodium bicarbonate, pH 8.0. An appropriate amount of 5 mg/ml reactive dye solution in Me₂SO was added to the mixture. The amount of dye was calculated according to the following formula: µl of dye stock solution = (mg/ml protein × 0.2 ml × MW_{reactive dye} × 200 × MR)/MW_protein. Where 200 is a unit conversion factor, and MR is the molar ratio of dye to protein in the reaction mixture. The reaction was stirred in the dark for 1 h and stopped by the addition of 5.5 µl of hydroxylamine provided with the kit and additional stirring for 15 min. Labeled antibodies were purified using spin columns provided with the kit. The degree of labeling was determined by measuring protein and dye concentrations (extinction coefficients provided by Molecular Probes) in a spectrophotometer and calculating protein/dye ratio. Typical labeling reaction resulted in 5-10 molecules of dye per one bivalent antibody molecule.

Direct Immunofluorescence Experiments-- BAC1.2F5 cells (32) and HeLa cells (33) were cultured as described previously. Cells were grown in 4-chamber LabTek^TM II Chamber Slides^TM. The cells were rinsed twice with PBS, fixed, and permeabilized. Six different fixation and permeabilization procedures were investigated to evaluate the reproducibility of staining patterns with Oregon Green^TM-labeled anti-CCT antibodies. The procedure of choice entailed fixation in 3.7% formaldehyde for 20 min at 25 °C followed by washing with PBS and permeabilization with 0.2% Triton X-100 for 10 min at 25 °C. The other five methods that were tested included: 1) fixation in 3.6% formaldehyde for 10 min at 25 °C and permeabilization with methanol:acetone (1:1) for 5 min at 25 °C (15); 2) fixation in 3.7% formaldehyde for 20 min and permeabilization with cold acetone for 20 min at 20 °C (3); 3) fixation and permeabilization in cold methanol for 6 min at 20 °C; 4) fixation and permeabilization in methanol:acetone (1:1) for 20 min at 4 °C; 5) fixation and permeabilization with 70% ethanol in 50 mM glycine for 15 min at 20 °C. All of the procedures, except the last one, resulted in the distribution of anti-CCT staining in both nuclear and cytoplasmic compartments. Using the last fixation/permeabilization procedure, anti-CCT was found in the cytoplasmic compartment only. After fixation and permeabilization, the cells were subjected to 3 × 5-min washes with PBS containing 1% dry milk and nonspecific binding was blocked with PBS with 1% dry milk for 1 h. Cells were then washed 3 × 5 min with PBS and treated with 1:50 dilution in PBS of the appropriate antibody. For antibody specificity controls, CCT antibodies were incubated for 1 h in the cold room rotator with a 20-fold molar excess of peptide before application to the cells. The antibody treatment was followed by 5 × 10-min washes with PBS with continuous shaking at 25 °C. For the colocalization studies, cells were treated with 50 µg/ml concanavalin A conjugates, 50 µg/ml wheat germ agglutinin conjugates, or a 1:100 dilution of fluorophore-labeled anti-protein disulfide isomerase for 1 h at 25 °C after the treatment with the anti-CCT antibody and washed an additional 5 × 5 min with PBS with shaking. Slides were mounted with Prolong^TM antifade in the mounting medium and covered with coverslips. For the localization of the nucleus, cells were treated with 1 µg/ml Hoechst 33258 dye.

Fluorescent antibodies and conjugates were visualized using a Leica DM IRBE laser scanning confocal microscope equipped with the TCS-NT scanning laser. The pictures were taken using Leica TCS-NT computer software. For high-resolution pictures, the images were digitally zoomed to bring a single cell into the field of view. Oregon Green^TM 488 fluorophore was visualized using an argon-ion laser and a fluorescein isothiocyanate filter set (488, 514 nm); Texas Red^TM was visualized with the krypton-ion laser using a tetramethylrhodamine isothiocyanate filter set (568, 647 nm); colocalization studies were conducted with argon and krypton lasers and double fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate filter sets. Hoechst 33258 dye was visualized with the UV laser and 352/461 nm filter set.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification and Sequence of the CCT2 cDNA-- Two human cDNA clones similar to CCT1 were identified (GenBank^TM accession numbers AA683266 and AI041180) using a BLAST search of the public expressed sequence-tagged data base of the National Center for Biotechnology Information. The DNA sequences were verified/corrected and completed, and analysis of the sequence information revealed the existence of a unique CCT mRNA, called CCT2, that was identical to CCT1 at the 5' end of the open reading frame but was predicted to encode a protein with a very different carboxyl terminus (Fig. 1). The cDNA sequence of clone AI041180 included both carboxyl termini representing the two variants of CCT with two in-frame stop codons to terminate translation, as well as the entire 5' coding sequence. These data indicated that two transcripts were expressed from the same gene and also indicated that the exon encoding the 2 carboxyl terminus precedes the one encoding the 1 terminus in the genomic structure. The sequence analysis indicated that CCT2 is a splice variant of CCT1. The cDNA for CCT2 was assembled to exclude the possibility of expression of CCT1, and subcloned into the expression vector pcDNA3.

View larger version (79K): [in this window] [in a new window]   Fig. 1.   Comparison of the cDNA sequences of CCT2, CCT1, and CCT. The human CCT2 cDNA sequence determined in this paper (GenBankTM accession number AF148464) was compared with the published cDNA sequences of human CCT1 (GenBankTM accession number AF052510) or human CCT (GenBankTM accession number L28957). Identical bases are boxed.

CCT Isoform Protein Sequence Comparison-- The predicted amino acid sequence of CCT2 was aligned with the sequences of CCT and CCT1 (Fig. 2). The predicted CCT2 protein had 369 amino acids and was identical to CCT1 from amino acids 1 to 320. After residue 320 there were 39 additional amino acids, including two groups of 5 and 4 amino acids (SSPTR, residues 321-325, and RSPS, residues 328-331), respectively, which were identical to sequences in CCT and missing from CCT1. The carboxyl terminus of CCT2 had 21 potential phosphorylation sites after position 310, including 19 serines and 2 threonine residues. As shown in Fig. 2 only 9 serines and 1 threonine of CCT2 align with the corresponding residues of CCT.

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Comparison of the predicted amino acid sequences of CCT, CCT1, and CCT2. The human CCT sequence (41) is compared with the CCT1 (3) and CCT2 (this study) sequences. The identical amino acid residues found in all three CCT isoforms are boxed.

Phosphorylation of CCT Isoforms-- The existence of 21 potential serine and threonine phosphorylation sites in the predicted carboxyl-terminal domain of CCT2 suggested that this enzyme was phosphorylated similar to the modification of CCT protein. This point was tested by transfecting COS-7 cells with CCT1, CCT2, or a vector control and followed labeling 48 h later with [³²P]orthophosphate (160 µCi/ml) for 60 min. Both CCT isoforms were immunoprecipitated with the amino-terminal anti-CCT antibody (B2 epitope), fractionated by SDS-PAGE, and the radiolabeled proteins were visualized by autoradiography. CCT2 was highly phosphorylated (Fig. 3) confirming the prediction made from the analysis of the primary structure of its carboxyl terminus. CCT1 was also phosphorylated, although to a signficantly lesser extent as was predicted from the fact that CCT1 had only 3 potential phosphorylation sites after amino acid 310. These data are consistent with the idea that the carboxyl-terminal domains of CCT1 and CCT2 were the exclusive sites of phosphorylation, as was shown with CCT (9, 16).

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Phosphorylation of CCT isoforms. COS-7 cells were transfected with pcDNA3 vector alone or pcDNA3 vectors carrying cDNAs encoding CCT1 or CCT2. After transfection cells were labeled with 1.6 mCi/dish of [32P]orthophosphate for 60 min, lysed on the dish, and immunoprecipitated with the anti-CCT antibody (B2 epitope) as described under "Experimental Procedures." Phosphorylated proteins were separated by SDS-PAGE on 12% gel and the bands visualized by autoradiography. The film was exposed for 48 h.

Expression and Amino-terminal Modification of CCT Isoforms-- In our previous report (3) describing CCT1 we suggested that the CCT1 protein was modified when expressed in COS-7 cells. Two immunoreactive proteins with apparent molecular masses of approximately 40 or 35 kDa were identified using anti-CCT (epitope B1) following transfection of COS-7 cells with CCT1 cDNA. The faster migrating protein species co-migrated with the major [³⁵S]methionine-labeled product of an in vitro transcription/translation reaction using the CCT1 cDNA as template. We proposed that the slower migrating CCT1 form identified in COS-7 cells may result from post-translational modification. We therefore attempted to obtain evidence of possible glycosylation, acylation or ubiquitination of the CCT1 protein, however, our efforts to identify the biochemical nature of the putative modification were unsuccessful. Experiments with a different lot of the commercial preparation of reticulocyte lysate also yielded two CCT1 translation products in vitro (Fig. 4), rather than the single product that was originally described (3), and addition of microsomes to the lysate did not alter the relative amounts of the two radiolabeled protein products.

View larger version (51K): [in this window] [in a new window]   Fig. 4.   CCT amino terminus is not modified in vivo. Left four lanes, transcription and translation in vitro of CCT1, CCT1[1-26], and CCT1[M27A] cDNAs was performed using a reticulocyte lysate system (Promega) containing 40 µCi/50 µl of [35S]methionine (1000 Ci/mmol). The products were separated by SDS-PAGE on 12% gel and the bands were visualized by autoradiography. Right four lanes, cellular expression of CCT1, CCT1[1-26], and CCT1[M27A] protein species. COS-7 cells were transfected with pcDNA3 plasmids carrying CCT1, CCT1[1-26], and CCT1[M27A] cDNAs and cell lysates were analyzed by immunoblotting 48 h later. Samples (50 µg of protein) of the total cell lysates were probed with anti-CCT antibody (B2 epitope) as described under "Experimental Procedures." Control samples were obtained using the pcDNA3 vector without a cDNA insert for expression experiments both in vitro and in vivo. The results are representative of three independent experiments.

Therefore, we tested the hypothesis that the faster migrating protein produced in the in vitro transcription at translation originated from an alternative translational initiation at methionine 27, since this second predicted methionine in the CCT open reading frame was within a favorable Kozak consensus context (34). Methionine 27 was changed to alanine by mutagenesis of the CCT1 cDNA and the derived expression construct, plasmid pPJ82, was used as a template for in vitro expression. CCT1(M27A) protein migrated at the same position as the "modified" CCT1 expressed in cells (Fig. 4). These data indicated that the initiation site in vivo was the first methionine in the open reading frame and also identified the correct mobility in SDS-PAGE for the full-length protein. On the other hand, expression of CCT1[1-26] in which the first 26 amino acids were deleted from the amino terminus yielded a protein that co-migrated with the "unmodified" or faster-migrating CCT1 expressed in cells (Fig. 4), and the major band produced by the in vitro transcription/translation system. These data showed the correct mobility for a protein that was 26 amino acids smaller corresponding to a CCT1 protein initiating at methionine 27 in the open reading frame. Thus, CCT was not post-translationally modified at the amino terminus when expressed in a cellular context and the differences in the in vivo and in vitro results was due to the artifactual initiation at an alternative methionine in the in vitro experiments.

Tissue-specific Expression of CCT Isoforms-- The tissue distribution and indication of the relative abundance of the CCT mRNAs was addressed by RT-PCR in a series of human tissues. The forward primer for the detection of both CCT isoforms was complementary to sequence within the 5' coding region of CCT and a sequence-specific reverse primer corresponded to the 3' ends of each of the two coding sequences for CCT1 and CCT2. The anticipated size of the CCT1 product was 256 bp whereas the CCT2 product was predicted to be 586 bp. The CCT primers were predicted to yield a PCR product 345 bp long. We incubated the CCT primers with the CCT purified cDNA as template, and conversely, the CCT primers were incubated with the CCT2 purified cDNA as template to verify the specificity of the primers under the thermocycling conditions. In both cases no DNA products were detectable (data not shown).

The data indicated that CCT was expressed in all tissues approximately at the same levels (Fig. 5). In contrast, the expression of the CCT isoforms differed among the tissues tested. Both isoforms of CCT were expressed in brain, with CCT2 being predominant. Liver also expressed both isoforms, with CCT1 giving a stronger signal. Placental tissue contained CCT1 transcripts with no detectable signal for CCT2. On the other hand, CCT2 was the predominant isoform expressed in HeLa cells whereas lower amounts of CCT1 were detected. An interesting variation in the development of lung tissue was suggested in that CCT and both CCT isoforms were expressed in fetal lung whereas mRNA from adult lung did not yield a signal for CCT and only CCT was expressed. These data are consistent with the results from Post's lab (35) where cDNAs encoding only CCT were cloned from an adult lung library.

View larger version (40K): [in this window] [in a new window]   Fig. 5.   Detection of CCT isoform expression by RT-PCR. RT-PCR was used to amplify CCT, CCT1, and CCT2 sequences from poly(A)+ RNA isolated from the indicated tissues. The position of the selected primers predicts the formation of products with the following sizes: CCT, 345 bp; CCT1, 256 bp; and CCT2, 586 bp. The DNA products were separated in a 1.3% agarose gel and visualized with ethidium bromide staining under UV light The above picture is representative of two separate experiments.

Subcellular Localization of CCT Isoforms-- We have developed two new antibodies, anti-CCT (B2 epitope) and anti-CCT2 (B3 epitope), to study the subcellular localization of the CCT isoforms. Immunoblotting data were obtained to confirm the specificity of the antibodies, following expression in COS-7 cells (Fig. 6). The anti-CCT antibody (B2 epitope) was directed against the amino terminus of CCT and recognized both the CCT1 and CCT2 isoforms on immunoblots as predicted. The anti-CCT2 antibody (B3 epitope) was directed against the unique carboxyl terminus of CCT2 and reacted only with the CCT2 isoform. The anti-CCT antibody reacted only with the CCT isoform.

View larger version (61K): [in this window] [in a new window]   Fig. 6.   Specificity of isoform-specific anti-CCT antibodies. COS-7 cells were transfected with pcDNA3 vector alone or pcDNA3 vectors carrying cDNAs encoding CCT, CCT1, or CCT2. Cell lysates were analyzed by immunoblotting 48 h later. Samples (50 µg of protein) of the total cell lysates were probed with anti-CCT, anti-CCT (B2 epitope), or anti-CCT2 (B3 epitope) antibodies as described under "Experimental Procedures".

CCT overexpressed in CHO58 cells was found to be localized mainly in the cell nucleus using a specific peptide antibody directed against the amino terminus of the protein (15, 36). On the other hand, CCT was also found to be both a nuclear and cytoplasmic protein in primary hepatocytes using an antibody that recognized the membrane interaction domain of CCT and that could potentially cross-react with CCT (37). In our previous report (3) we showed that CCT1 was an extranuclear protein using a specific anti-CCT amino-terminal antibody (B1 epitope). The above studies utilized conventional immunofluorescent microscopy to visualize the CCT proteins. In the present report, we used confocal microscopy to investigate the cellular localization of the  isoforms in more detail. Confocal microscopy was advantageous because it detected proteins at the same focal plane, thoroughly increasing the resolution of cellular structures compared with previously used techniques. We also coupled the fluorescent dyes directly to the affinity-purified primary antibodies at a high molar ratio (5-10 mol of dye/mol of bivalent antibody), thus increasing the sensitivity of detection of endogenously expressed protein. We compared the distribution of CCT2 in cells with the distribution of the two other CCT isoforms, CCT and CCT1, using direct immunofluorescence microscopy with confocal imaging and affinity-purified isoform-specific antibodies.

All of the CCT antipeptide antibodies used were raised in rabbits and direct coupling of different dyes to the antibodies also allowed the co-visualization of the CCT isoforms in the same in situ context. Fixation and permeabilization conditions were optimized as described under "Experimental Procedures" and the conditions (3.7% formaldehyde, 0.2% Triton) were chosen on the basis of reproducibility, consistency with the other methods, and preservation of morphology. An antibody dilution series was performed following each coupling reaction and cellular fluorescence patterns were recorded using antibody preparations at as high a dilution as possible to minimize possible nonspecific detection of unrelated proteins. The specificity of the fluorescent signal in cells was confirmed by preincubation of the antibodies with the corresponding peptide epitopes (Fig. 7). Multiple CCT antibodies (B1, B2, and B3 epitopes) were used to confirm the results for CCT. The CCT proteins from both human and rodent species are known to be identical at the amino terminus (38-41) and the mouse and human CCT proteins are also identical at the amino terminus.²

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Specificity of the fluorescent detection of CCT isoforms in cells. Three upper panels, staining of BAC1.2F5 cells with Oregon GreenTM-labeled anti-CCT, Texas RedTM-labeled anti-CCT (B2 epitope), or Texas RedTM-labeled anti-CCT2 (B3 epitope) antibodies was performed. The images were acquired with a × 63 objective and digitally zoomed. Three lower panels, Oregon GreenTM-labeled anti-CCT, Texas RedTM-labeled anti-CCT (B2 epitope), and Texas RedTM-labeled anti-CCT2 (B3 epitope) antibodies were preincubated with 1 mM of the corresponding antigenic peptides before staining the cells. The images were acquired with the × 40 objective at the maximum intensity of the lasers.

The CCT and - isoforms were visualized in several different cell types, including BAC1.2F5 murine macrophage cells, HeLa human carcinoma cells, and the CHO58 hamster ovary cells (Fig. 8A). In all three cell lines, CCT protein was largely found in the nucleus but a significant signal was also detected outside of the nucleus. The extranuclear CCT co-localized with concanavalin A (Fig. 8B), an agglutinin with a high affinity for mannose residues and a marker for the ER (42), as determined by computer-mediated overlay of the two distinct fluorescent images. Since there was only one antibody specific for CCT, a cDNA encoding an FLAG epitope-tagged CCT was also transfected into CHO58 cells and localized with anti-FLAG antibody (M2 antibody) to confirm results obtained with the anti-CCT antibody and ensure that an unrelated protein did not possess the same peptide epitope (Fig. 8C). CCT2 protein was also found in the three cell lines and was situated outside of the nucleus (Fig. 8A). Antibodies that recognized both CCT isoforms (B1 and B2 epitopes) and those that were specific for CCT2 co-localized not only with concanavalin A but also with anti-protein disulfide isomerase (Fig. 9), another marker protein for the ER organelle (43). Antibodies for CCT and CCT2 also co-localized with each other (Fig. 10). Neither the CCT nor CCT antibodies associated to a high degree with the Golgi bodies as determined by co-staining with fluorescently tagged wheat germ agglutinin (data not shown), a marker for the Golgi organelle (44). These data support the conclusion that CCT was found both in the nucleus and associated with the ER and that the CCT isoforms were associated with the ER. Direct evidence of CCT2 expression and ER association was obtained with this approach but the specific occurrence of CCT1 could not be determined with these immunological reagents.

View larger version (42K): [in this window] [in a new window]   Fig. 8.   CCT and CCT isoforms in three cell types. A, murine macrophage (BAC1.2F5) cells, human carcinoma (HeLa) cells, or Chinese hamster ovary fibroblast (CHO) cells were stained with Oregon GreenTM-labeled anti-CCT (three upper panels) or Texas RedTM-labeled anti-CCT2 (B3 epitope) antibodies (three lower panels). Images were acquired with the × 63 objective and digitally zoomed. B, BAC1.2F5 cells were co-stained with Oregon GreenTM-labeled anti-CCT antibodies and Texas RedTM-labeled concanavalin A. The images were acquired with the × 63 objective and digitally zoomed to bring the single cell into the field of view. Two image files were obtained with different filter sets and the files were overlaid by computer (third panel). The orange color indicates co-localization. C, CHO cells were transfected with cDNA encoding FLAG-CCT and co-stained with Texas RedTM-labeled anti-FLAG antibody and Oregon GreenTM-labeled anti-CCT antibody. Images were acquired with the × 63 objective and digitally zoomed. The overlay of the two image files obtained with different filter sets was computer-mediated (third panel). The orange color indicates region of co-localization.

View larger version (58K): [in this window] [in a new window]   Fig. 9.   CCT2 associates with the ER. BAC1.2F5 cells were co-stained with Texas RedTM-labeled anti-CCT (B2 epitope), and Oregon GreenTM-labeled concanavalin A (first row); co-stained with Texas RedTM-labeled anti-CCT (B2 epitope) antibodies and Oregon GreenTM-labeled anti-mouse protein disulfide isomerase antibodies (second row); or Texas RedTM-labeled anti-CCT2 (B3 epitope) antibodies and Oregon GreenTM-labeled anti-CCT (B2 epitope) antibodies (third row). The images were acquired with the × 63 objective and digitally zoomed to bring the single cell into the field of view. The computer overlays of the image files are shown in the far left column of each row.

View larger version (16K): [in this window] [in a new window]   Fig. 10.   CCT2 expression in COS-7 cells. CCT specific activity was determined in COS-7 cells transfected with plasmids expressing CCT2 (pAL2) () or an empty vector control (pcDNA3) (). The cells were harvested, extracts were prepared and assayed for CCT activity 48 h after transfection as described under "Experimental Procedures." The results are representative of duplicate experiments.

Overexpression of CCT2 in COS-7 Cells-- The similarities among amino acid sequences of CCT2, CCT1, and CCT suggested that CCT2 would also exhibit CCT enzyme activity. Transfection of COS-7 cells with plasmid pAL2 containing the CCT2 cDNA resulted in significantly increased CCT enzyme specific activity (Fig. 10), from 2.5 to 27 nmol/min/mg, in the crude cell lysates. Overexpression of CCT2 activity also resulted in an increased incorporation of [methyl-³H]choline into cellular CDP-choline, PtdCho, and glycerophosphocholine (data not shown) comparable to the levels of radioactive metabolites following overexpression of CCT1 in COS-7 cells (3).

CCT Rescued CHO58 Cells-- The CHO58 cell line is conditionally defective for CCT activity (28) and cannot synthesize sufficient PtdCho to support growth at 40 °C. Transfection of CHO58 cells with a CCT cDNA complements the defective CCT activity and the overexpression of the  isoform supported the proliferation of CHO58 cell colonies after shifting the cultures to the restrictive temperature (45). The differences in primary structure and subcellular localization between CCT and CCT2 raised a question as to whether CCT2 had a cellular function similar to that of CCT. To address this issue, CHO58 cells were transiently transfected with cDNAs encoding CCT1, CCT2, and CCT as a positive control, or vector alone as a negative control (Fig. 11). After 72 h at 40 °C, 7 colonies remained, in the control dishes transfected with vector alone, indicating that reversion of the background genetic phenotype did not occur under these experimental conditions. In contrast, the dishes transfected with CCT, CCT1, or CCT2 cDNAs, hundreds of colonies were evident, indicating that overexpression of any of the CCT isoforms could complement the CCT defect in the CHO58 cells. These data suggest that CCT, CCT1, and CCT2 perform equivalent biochemical functions.

View larger version (70K): [in this window] [in a new window]   Fig. 11.   CCT1 and CCT2 can rescue CHO58 cells. Cells grown at 33 °C were transfected with cDNAs encoding CCT, CCT1, CCT2, or pcDNA3 vector alone. Cells were transferred to 40 °C 24 h after transfection and cultured at 40 °C for an additional 72 h. Dishes were then washed twice with phosphate-buffered saline and adherent colonies were stained with Coomassie Blue R-250 as described under "Experimental Procedures."

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A major finding of this study is the identification of the CCT2 cDNA and the characterization of the protein. CCT, CCT1, and CCT2 have very similar catalytic and amphipathic helical domains consistent with their stimulation by lipid regulators (1-3). Also, both the  (6) and  isoforms are inhibited by antineoplastic phospholipids as would be predicted from the similarity of their primary sequences and the metabolic redistribution of the PtdCho precursors in drug-treated cells that express all isoforms (i.e. BAC1.2F5 (6) and HeLa cells (33)). CCT1 and CCT2 likely arise from alternate splicing of the CCT mRNA which results in the production of two mRNAs that encode proteins that differ only at their carboxyl terminus. CCT1 is a protein of 330 amino acids whereas CCT2 has 369 amino acids. The additional 39 carboxyl-terminal residues in CCT2 closely resembles the carboxyl-terminal phosphorylation domain of CCT. Within this domain, CCT2 has 22 potential phosphorylation sites (19 serines and 2 threonines) compared with the 13 serine residues known to be phosphorylated in the carboxyl-terminal domain of CCT (16). Accordingly, CCT2, like CCT, is extensively phosphorylated in vivo. CCT1 lacks the numerous phosphorylation sites present in CCT and CCT2 and is phosphorylated to a minor extent in vivo indicating that CCT1 may not be subject to regulation by protein kinases. After the splice junction at amino acid 323, CCT2 has two regions of five (SSPTR) and four (RSPS) residues identical to sequences known to be phosphorylated in CCT (16). Also, CCT2 contains a unique sequence, SSPTRSRSPSRSP, containing the RSPXR motif similar to the one found in neurofilament H (KSPXK) that specifies phosphorylation by cyclin-dependent kinase 5 (46). Phosphorylation attenuates CCT biochemical activity by interfering with lipid stimulation (17) and unphosphorylated CCT exhibits a higher degree of membrane association in cells (24). Phosphorylation is predicted to exert the same regulatory influence on CCT2 as on CCT.

CCT1 was proposed to be post-translationally modified following overexpression in COS-7 cells, resulting in slower migration during SDS-PAGE (3). Truncation of the amino-terminal 26 amino acids resulted in a protein that co-migrated slightly faster than the full-length product of in vitro transcription/translation of the CCT1 cDNA (3). Examination of new data that was obtained during comparison with the most recently discovered isoform, CCT2, revealed that the faster migrating product of the in vitro transcription/translation was an artifact where translation was initiating at Met-27. CCT1 and CCT2 proteins initiated at Met-1 when expressed in vivo as demonstrated by interaction with the antibody specific for residues 5 through 22 (B2 epitope) and confirmed by co-migration with the M27A mutant (Fig. 4).

CCT1 and CCT2 are distinguished from CCT by their selective localization to the ER suggesting that the  isoform plays a special role in PtdCho metabolism in the ER compartment. Our use of laser-scanning confocal microscopy and direct labeling of the primary antibodies with fluorescent tags resulted in improved resolution of cellular structures and a more sensitive detection of endogenous CCT isoforms than in previous studies. Computer overlays of the immunofluorescent images confirm that both CCT isoforms colocalize with each other and with ER-specific markers. Although, significant amounts of CCT are found distributed throughout the interphase nucleus, except the nucleolus, CCT also colocalizes with ER-specific markers. These results are in general agreement with the available information from other laboratories. An investigation of the cellular distribution of CCT in hepatocytes using immunoelectron microscopy and an antibody that was potentially cross-reactive with CCT localized CCT to both the nuclear and extranuclear compartments (37). CCT was assigned to both the cytoplasm and the ER, however, membranous structures were not distinct in the images used as evidence for cytoplasmic localization, probably due to the limitations of the fixation procedure. It is clear from our images (Fig. 8) that there is very little extranuclear CCT that is not associated with the ER. Indirect immunofluorescence employed by the Kent group (15, 21, 22, 36, 47) showed CCT to predominately reside in the nucleus. Although there is an indication of cytoplasmic fluorescence in some of their images, the bright nuclei coupled with the inability of this technique to examine thin sections through the cells may have obscured extranuclear CCT. This may be particularly relevant in their experiments using CHO58 cells to localize overexpressed CCT (15, 36, 40) since it is possible that the number of CCT sites on the ER is limiting and that supraphysiological concentrations of CCT accumulate in the nucleus. Nuclear CCT is the likely source of the soluble CCT pool defined by subcellular fractionation and digitonin permeabilization experiments (1) since the remaining CCT staining in cells is associated with membrane systems. CCT is primarily regulated by the membrane lipid environment (1, 2) and localization of CCT to the ER places the enzyme in a prime position to respond to changes in the bulk membrane environment to maintain homeostasis.

All CCT isoforms have the same biochemical function and accelerate PtdCho synthesis when overexpressed in cells (Fig. 10) (3, 26). Also, CCT, CCT1, or CCT2 can supply the enzymatic activity necessary to support the growth of a cell line conditionally defective in CCT activity. We detected both CCT and CCT proteins in the mutant CHO58 cell line (Fig. 8A) indicating that both proteins may be conditionally defective to obtain the temperature-sensitive defect in PtdCho biosynthesis (28). The ability of either CCT or CCT to complement the temperature-sensitive phenotype suggests that reversion at either the CCT or CCT genetic locus could give rise to a temperature-resistant derivative cell line (48). However, any conclusions reached on the basis of expression studies in CHO58 cells must be tempered by the understanding that overexpression studies are a very blunt experimental tool to address the functionality of isoforms or mutants. Catalytically compromised or mislocalized proteins can complement mutant phenotypes if the defective proteins are expressed at a high enough level. Supraphysiological concentrations of CCT swamp the cells with CDP-Cho and trigger a compensatory response to the overproduction of PtdCho by enhancing degradation (3, 26, 27). Thus, enforced CCT may complement a CCT function(s) by swamping the cells with CDP-Cho. Also, CCT proteins defective in catalytic activity, regulatory function, or cellular localization may complement the CHO58 cells when overexpressed, whereas they may not be able to sustain growth if present as a single copy. The definition of the specific functions of the CCT isoforms and the importance of nuclear versus ER localization await more detailed genetic experiments.

A specific cellular function for CCT remains speculative, although analysis of the tissue-specific distribution of CCT isoforms does suggest some hypotheses. CCT2 could play an important role in neuronal development and function since the brain has the highest levels of CCT2 expression (Fig. 5) and PtdCho biosynthesis is critical to axons (49). The finding of CCT associated with the ER suggests that this isoform may be involved in tissues that secrete PtdCho. For example, CCT1 is highly expressed in placenta (Fig. 5) and may play a role in PtdCho bioysnthesis in this lipogenic tissue which secretes and supplies phospholipid to a developing embryo (50). Liver and fetal lung also express both CCT isoforms, but CCT1 is predominant (Fig. 5). The absence of CCT expression in adult lung (Fig. 5) does not fit with this hypothesis since PtdCho biosynthesis plays an important role in surfactant secretion and pulmonary function. A precise determination of a function for CCT will require the analysis of genetically engineered animals that do not express this isoform.

	ACKNOWLEDGEMENT

We thank Jina Wang for excellent technical assistance.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM 45737, Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities.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.

^§ Present address. Roche Diagnostics Systems, 1080 US Highway 202, Somerville, NJ 08876-3771.

To whom correspondence should be addressed: Biochemistry Department, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. Tel.: 901-495-3494; Fax: 901-525-8025; E-mail: suzanne.jackowski@stjude.org.

² M. Kavim and S. Jackowski, unpublished data.

	ABBREVIATIONS

The abbreviations used are: PtdCho, phosphatidylcholine; CCT, CTP:phosphochoine cytidylyltransferase; CDP-Cho, cytidine diphosphocholine; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair; PBS, phosphate-buffered saline. CHO, Chinese hamster ovary; ER, endoplasmic reticulum; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)- propane-1,3-diol; PAGE, polyacrylamide gel electrophoresis.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTA