Cloning and Characterization of a Second Human CTP:Phosphocholine Cytidylyltransferase*

Athanasios LykidisDagger , K. Gopal Murti§, and Suzanne JackowskiDagger parallel **

From the Departments of Dagger  Biochemistry and § Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105 and the Departments of parallel  Biochemistry and  Pathology, University of Tennessee, Memphis, Tennessee 38163

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

CTP:phosphocholine cytidylyltransferase (CCT) is a key regulator of phosphatidylcholine biosynthesis, and only a single isoform of this enzyme, CCTalpha , is known. We identified and sequenced a human cDNA that encoded a distinct CCT isoform, called CCTbeta , that is derived from a gene different from that encoding CCTalpha . CCTbeta transcripts were detected in human adult and fetal tissues, and very high transcript levels were found in placenta and testis. CCTbeta and CCTalpha proteins share highly related, but not identical, catalytic domains followed by three amphipathic helical repeats. Like CCTalpha , CCTbeta required the presence of lipid regulators for maximum catalytic activity. The amino terminus of CCTbeta bears no resemblance to the amino terminus of CCTalpha , and CCTbeta protein was localized to the cytoplasm as detected by indirect immunofluorescent microscopy. Whereas CCTalpha activity is regulated by reversible phosphorylation, CCTbeta lacks most of the corresponding carboxyl-terminal domain and contained only 3 potential phosphorylation sites of the 16 identified in CCTalpha . Transfection of COS-7 cells with a CCTbeta expression construct led to the overexpression of CCT activity, the accumulation of cellular CDP-choline, and enhanced radiolabeling of phosphatidylcholine. CCTbeta protein was posttranslationally modified in COS-7 cells, resulting in slower migration during polyacrylamide gel electrophoresis. Expression of CCTbeta /CCTalpha chimeric proteins showed that the amino-terminal portion of CCTbeta was required for posttranslational modification. These data demonstrate that a second, distinct CCT enzyme is expressed in human tissues and provides another mechanism by which cells regulate phosphatidylcholine production.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

CCT1 is a key enzyme in the regulation of PtdCho biosynthesis, and the mechanisms that regulate its expression, activity, and subcellular localization are the focus of current research (for reviews, see Refs. 1 and 2). cDNAs that encode CCTalpha proteins have been identified and sequenced in rat (3), hamster (4), mouse (5), and human (6), and there are only minor differences among these mammalian cDNAs (see Ref. 6 for a comparison). Their catalytic properties are thought to be essentially identical, and CCTalpha can be divided into four distinct functional domains (see Fig. 1). The amino-terminal domain between residues 1 and 71 contains a sequence that specifies the nuclear localization of the protein between residues 2 and 28 (7, 8). The catalytic core extends from residues 72 to 233. This region of the protein is conserved from yeast to mammals and is responsible for substrate binding and catalysis. In particular, the conserved HXGH motif is essential for cytidylyltransferase activity (9, 10). The third domain, located between residues 256 and 288, contains three 11-residue amphipathic repeats that form alpha -helices following association with lipid regulators and contribute to the reversible membrane association of the enzyme (11-15). The binding of stimulatory lipids to this region greatly enhances catalytic activity by lowering the Km of the enzyme for CTP into the range corresponding to cellular concentrations of the nucleotide (16). CCTalpha is also negatively regulated by lipids and is potently inhibited by sphingosine (17), lysophosphatidylcholine (18), and antineoplastic phospholipids (18, 19). The fourth domain of CCTalpha is the carboxyl-terminal phosphorylation domain between residues 315 and 367. CCTalpha membrane association and activity are modulated by reversible phosphorylation (20, 21), and all of the phosphorylation sites are located in the carboxyl-terminal region (22). Phosphorylation attenuates CCTalpha biochemical activity by interfering with lipid stimulation (23), and unphosphorylated CCTalpha exhibits a greater degree of membrane association in cells (20).

CCTalpha has been localized using cellular in situ methods to the nucleus in Chinese hamster ovary cells (7, 8), but in rat hepatocytes, the protein has been detected in both the nuclear and cytoplasmic compartments (24). CCTalpha has also been identified in association with Golgi membranes (25, 26), endoplasmic reticulum, and transport vesicles (27) using biochemical methods. There is only one isoform of CCT expressed in yeast (28), and only one isoform of CCT (CCTalpha ) has been identified, purified, or cloned from mammalian sources (2). The existence of a conditionally lethal Chinese hamster ovary cell mutant with a temperature-sensitive defect in CCTalpha activity (29) also suggested that there was only a single CCT isoform in mammalian cells. A single genetic locus for CCTalpha was identified on mouse chromosome 16 (5), and the murine CCTalpha gene has been cloned (30). In this work, we identify a unique, second human CCT isoform, called CCTbeta . CCTbeta catalyzes the same enzymatic reaction as CCTalpha and requires the presence of lipids for full activity. However, CCTbeta lacks the nuclear targeting sequence and the phosphorylation domain of CCTalpha , suggesting that CCTbeta is distinct from CCTalpha with regard to its subcellular localization and regulation.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Sources of supplies were as follows: American Radiolabel Company, Inc., phospho[methyl-14C]choline (specific activity, 55 mCi/mmol) and [methyl-3H]choline (specific activity, 85 mCi/mmol); Amersham Pharmacia Biotech [35S]methionine (specific activity, >1000 Ci/mmol); CLONTECH, human multiple tissue Northern blots; Life Technologies, Inc., LipofectAMINE reagent; Promega, restriction endonucleases and other molecular biology reagents; Invitrogen, pcDNA3 plasmid; FMC Corp., Sea-Kem, molecular biology grade agarose; American Type Culture Collection, cDNA clone AA382871; Sigma, CTP and buffers; Avanti Polar Lipids, PtdCho and fatty acids; Analtech, thin-layer chromatography plates. All other supplies were reagent grade or better.

Anti-CCTalpha rabbit polyclonal antiserum was raised against a synthetic peptide (MDAQSSAKVNSRKRRKE) corresponding to the first 17 amino acids of CCTalpha . AntiCCTbeta rabbit polyclonal antiserum was raised against a synthetic peptide (MEEIEHTCPQPRL) corresponding to amino acids 27-39 of CCTbeta . Peptides and peptide antigens were prepared by the Molecular Resource Center of St. Jude Children's Research Hospital. To prepare antigen, each peptide was coupled to keyhole limpet hemocyanin via an additional cysteine at the carboxyl terminus of the peptide. Immunization of rabbits and collection of antisera 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.

Isolation of the CCTbeta cDNA-- Human Genome Systems identified and provided a clone that exhibited significant sequence similarity to CCTalpha . The protein expressed from this cDNA, however, did not exhibit significant CCT catalytic activity. Sequence information from this clone was used to search the public expressed sequence tagged data base. We identified a clone (GenBankTM accession no. AA382871) that contained 40 bp of related sequence at the 5' end. We purchased this clone from American Type Culture Collection and sequenced the cDNA 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. The cDNA contained a single open reading frame we called CCTbeta . A 1.3-kb BamHI-XhoI fragment was excised and subcloned into the mammalian expression vector, pcDNA3, to generate plasmid pPJ34, which expressed CCTbeta from the constitutive cytomegalovirus promoter.

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.

Construction of Plasmids for Expression of CCT Chimeras and CCTbeta Amino-terminal Truncation-- Rodent CCTalpha (pWYCT) and human CCTbeta (pPJ34) cDNAs cloned into pcDNA3 were digested with SspI. The pcDNA3 vector has an SspI site distanced approximately 1 kb from the 5'-end of the T7 promoter. The CCTalpha cDNA has an SspI site at nucleotide 260, whereas CCTbeta has an SspI site at nucleotide 311. The fragments that contained either CCTalpha or CCTbeta sequence plus vector sequence were purified. The 1.3-kb fragment generated from CCTbeta cDNA that encoded the amino terminus was ligated to the 5-kb fragment of pWYCT to generate pCCTbeta /CCTalpha , and the 1.3-kb fragment from the CCTalpha cDNA encoding the amino terminus was ligated to the 5-kb fragment of pPJ34 to generate pCCTalpha /CCTbeta . The resulting plasmids were checked for correct orientation with the polymerase chain reaction using the T7 and SP6 primers of pcDNA3.

Plasmid pPJ34 was digested with NcoI, and the resulting 2185-bp fragment was purified. The DNA overhanging sequences were filled in with Klenow fragment and purified. The fragment was next digested with XhoI to yield a 1180-bp fragment, and after purification, this fragment was ligated into pcDNA3 that had been previously digested with EcoRV and XhoI, resulting in plasmid pPJ35. Inserts in pcDNA3 were screened by polymerase chain reaction using T7 and Sp6 primers. DNA sequencing confirmed the truncation of the first 26 amino acids of CCTbeta .

CCT Assay-- CCT activity was determined essentially as described previously (31). The standard assay contained 64 µM lipid activator (PtdCho:oleic acid, 1/1), 4 mM CTP, 10 mM MgCl2, 150 mM bis-Tris-HCl, pH 6.5, 1 mM phospho[14C]choline (specific activity, 4.5 mCi/mmol), in a final volume of 50 µl. The reaction mixtures were 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 co-migration with a standard, scraped from the plate, and quantitated by liquid scintillation counting. Protein was determined according to the Bradford method (32).

Isolation of CCTbeta from Endogenous Lipids-- CCTbeta was isolated from COS-7 cells 48 h after transfection with plasmid pPJ34. Cells were washed with phosphate-buffered saline and harvested by centrifugation, and the pellet was lysed by incubation in lysis buffer (10 mM NaCl, 1 mM EDTA, 2 mM dithiothreitol, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 50 mM NaF, 100 µM Na3VO4, 10 mM HEPES, pH 7.4) for 1 h on ice. The cells were disrupted by sonication, and the particulate matter was removed by centrifugation. The supernatant was loaded onto a 0.5-ml DEAE-Sepharose column and the column was washed with 1.5 ml of each of the following in succession: lysis buffer, lysis buffer plus 1% Nonidet P-40, lysis buffer, lysis buffer plus 0.25 M NaCl, lysis buffer plus 0.5 M NaCl, and lysis buffer plus 1.0 M NaCl. The eluant was collected in 0.5-ml fractions, and CCT activity was located in the 0.25 M NaCl wash. This procedure is essentially the same as described in previous papers (16, 23). CCTbeta activity that was eluted from the column could only be detected in the presence of added lipid activators.

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. Transfections using LipofectAMINE reagent were performed according to the manufacturer's instructions. Briefly, 10 µg of plasmid and 60 ml of LipofectAMINE reagent were separately diluted into 0.8 ml of serum-free medium. The two solutions were combined and incubated at 25 °C for 45 min. Next, 6.4 ml of serum-free medium was added to each tube, and the diluted solution was overlaid onto the COS-7 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, and the cells were incubated for an additional 24 h at 37 °C and then harvested for analysis.

Metabolic Labeling-- COS-7 cells were transfected with vectors expressing either CCTalpha , CCTbeta , or a control. The total plasmid amount in each of the transfections was 10 µg. At 24 h after transfection, the medium was changed, and the cells were labeled for an additional 24 h with [methyl-3H]choline (3 µCi/ml). Cells were washed three times with 10 ml of phosphate-buffered saline and harvested in 10 ml of the same buffer, and the cell pellets were extracted using 720 µl of chloroform/methanol/concentrated HCl (1:2:0.02, v/v). Next, 240 µl of chloroform and 240 µl of water were added, and following vortex mixing, the phases were separated by centrifugation. The radioactivity in the soluble and phospholipid phases was quantitated. Samples of the soluble phase were separated on Silica Gel G thin layers developed with 2% ammonium hydroxide/95% ethanol (1:1, v/v), and the organic phase was analyzed on Silica Gel 60 thin layers developed with chloroform/methanol/ammonium hydroxide (60:35:8, v/v). About 90% of the labeled material in the organic phase was PtdCho under these labeling conditions. Choline-derived metabolites were identified by co-migration with standards.

Northern Blots-- Three multiple human tissue Northern blots were purchased from CLONTECH and were hybridized and washed according to the manufacturer's instructions. The blots were first hybridized with 32P-labeled probe prepared from a 1.3-kb BamHI-XhoI fragment that covered the entire CCTbeta cDNA. The blots were then stripped and hybridized with a 32P-labeled probe prepared from the 582-bp SacI fragment of the human phosphatidylinositol synthase (pis1) cDNA (33). The blots were stripped again and hybridized with a 32P-labeled probe prepared from a 320-bp PstI-ApaI fragment representing the 3' region of the CCTbeta cDNA. This area of the CCTbeta cDNA did not share any sequences in common with CCTalpha .

Immunoblots-- 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 either purified anti-CCTalpha (1:200 dilution) or purified anti-CCTbeta (1:200 dilution) as the primary antibody. The Amersham Pharmacia Biotech ECL Western blotting reagents and protocol were used to identify the immunoreactive proteins.

Immunofluorescence Microscopy-- HeLa cells grown on coverslips were fixed with 3.7% paraformaldehyde, permeabilized with cold acetone, and processed as described (34). Affinity-purified anti-CCTbeta primary antibody was diluted in 0.15 M NaCl, 10 mM Tris-HCl, pH 8.0. The cells were incubated with anti-CCTbeta antibodies at increasing dilutions followed by fluorescein-conjugated secondary antibodies. The coverslips were mounted with p-phenyldiamine, the cells viewed in a Zeiss IM-35 microscope equipped with fluorescence optics, and photographs were made on Kodak Tri-X pan film. Controls from which the primary antibody was excluded showed no significant fluorescence. Preincubation of the primary antibody with the peptide did not yield significant fluorescence. Two other controls assured selective labeling of the nuclear and cytoplasmic compartments. Both a nucleolar marker (anti-p120 antibodies, Becton Dickinson) and a cytoplasmic (cytoskeletal) marker (anti-vimentin antibodies, Boehringer Mannheim) were used to label the cells to confirm appropriate staining of cellular compartments.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Identification of the CCTbeta Clone-- A BLAST search of the proprietary human expressed sequence tagged data base of Human Genome Sciences revealed the existence of a cDNA with considerable similarity to mammalian CCTs. However, the protein expressed by this cDNA was not catalytically active, indicating that it did not contain a complete CCT coding sequence. Sequence information from this clone was used to search the public expressed sequence tagged data base. We identified a second clone (GenBankTM accession no. AA382871) isolated from a human testis library that contained a 140-bp related sequence at the 5' end. This clone, called CCTbeta , was purchased from American Type Culture Collection. Both cDNA strands were sequenced, and the clone contained the entire CCTbeta coding sequence. The cDNA sequence of human CCTbeta was compared with the cDNA sequence of human CCTalpha (see Fig. 2). The analysis of the sequence (see below) indicated that the CCTbeta cDNA encoded a new CCT isoform.

Similarities and Differences between the Predicted Protein Sequences and the cDNAs of CCTalpha and CCTbeta -- The predicted amino acid sequences of human CCTalpha and CCTbeta are compared in Fig. 1. The catalytic cores of human CCTalpha and CCTbeta are nearly identical and extend from amino acids 72 through 233. The catalytic core in CCTbeta has 64% identity with the equivalent yeast CCT domain that is located between amino acids 99 and 260 of the yeast protein (28). Three of the amino acids in CCTbeta that are different from CCTalpha (N120K, V136L, and R162K) are identical to the yeast CCT sequence. Three other amino acids that are different in CCTbeta compared with CCTalpha (E126D, D134E, and E160K) are identical to the residues found in the catalytic core of the yeast MUQ1 sequence, which has been identified as phosphoethanolamine cytidylyltransferase (36). Also, the catalytic domains of human phosphoethanolamine cytidylyltransferase are highly related to the analogous domains in CCTalpha and CCTbeta (37). These sequence similarities strongly suggested that the CCTbeta cDNA encoded a protein with cytidylyltransferase activity.


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Fig. 1.   Domain structure and comparison of the predicted amino acid sequences of CCTalpha and CCTbeta . A, diagrammatic representation of the domain structure of CCTalpha compared with the predicted domain structure of CCTbeta . B, the identical amino acid residues found in both CCTalpha and CCTbeta are boxed.

The amino-terminal domain of CCTbeta is distinct from the amino-terminal region of human CCTalpha (Fig. 1). CCTbeta lacks the human CCTalpha sequence 8KVNaRKRRKEaPGPNGATEED28, which is postulated to mediate transit of the protein to the nuclear compartment. Amino acids 11 and 18 are designated in lowercase because they differ from the rodent CCTalpha sequence that is known to be the minimal protein sequence that is both necessary and sufficient for localization of rodent CCTalpha to the nucleus (8). The significant difference in the amino-terminal sequences of CCTalpha and CCTbeta and the lack of a discernible nuclear localization motif in CCTbeta suggested that CCTbeta would not be localized in the nucleus. On the other hand, the amino-terminal domain of CCTbeta has some identity to the yeast amino-terminal region (28) at amino acids 38 through 40 (RLT), and at Ala48, Thr51, and Asn52. These sequence similarities indicate that the amino terminus of CCTbeta is more related to the yeast CCT than mammalian CCTalpha ; however, the significance of this correlation is not obvious because a specific function for the amino terminus of yeast CCT has not been described.

The predicted CCTbeta protein sequence exhibits some significant similarities and distinct differences from the CCTalpha sequence in the amphipathic helical and phosphorylation regulatory domains (Fig. 1). The helical domain of human CCTbeta is highly related to the analogous domain in CCTalpha , with 88% amino acid identity between residues 256 and 288 and with conservative substitutions at K259R, Q265N, and K266R. This domain in rodent CCTalpha is required for the lipid-dependent decrease in the CTP Km associated with stimulation of CCT activity (16) and mediates the reversible binding of CCTalpha to phospholipid bilayers (11-15). A similar amphipathic helical domain is absent in the yeast CCT (28), although this enzyme is activated by lipids (38). The similarity in the helical regions of CCTalpha and CCTbeta suggested that CCTbeta activity would be stimulated by the interaction with lipids in a manner similar to mammalian CCTalpha and that CCTbeta would undergo reversible association with cellular membranes. Lipid stimulation of CCTalpha activity is regulated by phosphorylation of the carboxyl-terminal domain (23). Sixteen serine residues are located in the CCTalpha domain, which extends from amino acid 315 through 367, and all of these serines are phosphorylated to some extent (22). In contrast, the shorter carboxyl-terminal domain of CCTbeta contains only three possible serine phosphorylation sites (Ser315, Ser319, and Ser323). Ser315 and Ser319 are potential proline-directed phosphorylation sites analogous to Ser315 and Ser319 in CCTalpha . Thus, the opportunities for the regulation of CCTbeta activity by reversible phosphorylation are more restricted in CCTbeta than in CCTalpha .

The cDNA sequences of CCTalpha and CCTbeta are distinct but there are regions that have significant similarity (Fig. 2). The similarities are most pronounced in the catalytic core and amphipathic helix domains that are the most conserved peptide regions between CCTalpha and CCTbeta . However, there are many differences in the cDNA sequence in these regions, illustrating that CCTalpha and CCTbeta arise from the transcription of different genes and not by the alternative splicing of a single gene. The 5' and 3' regions of the cDNAs reflect the lack of similarity between the two proteins in the amino and carboxyl-terminal regions.


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Fig. 2.   A comparison of the cDNA sequences of CCTalpha and CCTbeta . The human CCTbeta cDNA sequence determined in this paper (GenBankTM accession no. AF052510) was compared with the published cDNA sequence of human CCTalpha (GenBankTM accession no. L28957). Identical bases are boxed.

Pattern of CCTbeta mRNA Expression-- The relative abundance of CCTbeta mRNA expression in a wide variety of human tissues was addressed by Northern blot analysis (Fig. 3). The blots were probed with the human phosphatidylinositol synthase (pis1) cDNA as a loading control. This enzyme in phosphatidylinositol biosynthesis is a "housekeeping" protein that is expressed at a relatively uniform level in human tissues (33). The blots were then probed with both the entire CCTbeta cDNA (Fig. 3) and with a 32P-labeled fragment from the 3' untranslated region of the CCTbeta cDNA. The pattern of expression was the same with both probes (not shown). Two sizes of CCTbeta transcripts were detected. The largest CCTbeta mRNA (~6.5 kb) was most abundant in brain, ovary, testis, and all fetal tissues examined. The second class of CCTbeta mRNAs were found between 1.1 and 1.9 kb. The 1.1-kb mRNA was detected in placenta, which was the most abundant source for CCTbeta mRNA in our survey. Testis also was an abundant source for CCTbeta transcripts, and two mRNAs of 1.6 and 1.9 kb were detected in this tissue. Although it is difficult to see in Fig. 3 due to the very high expression of CCTbeta in placenta and testis, CCTbeta mRNA species of either 1.1 or 1.9 kb were faintly detected in all tissues examined. Thus, CCTbeta mRNA is widely distributed in human tissues and expressed at very high levels in testis and placenta.


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Fig. 3.   Pattern of CCTbeta expression in human tissues. Three multiple human tissue Northern blots were purchased from CLONTECH and were hybridized and washed according to the manufacturer's instructions. The blots were first hybridized with a 32P-labeled probe prepared from a 1.3-kb BamHI-XhoI fragment that contained the entire human CCTbeta cDNA. The blots were then stripped and hybridized with a 32P-labeled probe prepared from the 582-bp SacI fragment of the human phosphatidylinositol synthase (Pis1) cDNA (33).

Substrate Specificity of CCTbeta -- The similarity of the catalytic core domains of CCTbeta and CCTalpha suggested that CCTbeta was a phosphocholine cytidylyltransferase. This prediction was confirmed by transfecting COS-7 cells with a CCTbeta expression construct (pPJ34) and measuring the CCT enzymatic activity in cell lysates (Fig. 4A). The introduction of the CCTbeta expression vector into the COS-7 cells led to a significant (8-fold) increase in the CCT specific activity from 4.8 to 38.4 nmol/min/mg of protein in cell extracts (Fig. 4A). These data establish that the CCTbeta cDNA encodes an active CTP:phosphocholine cytidylyltransferase. Alternative substrates for CCTbeta (phosphoethanolamine, glycerol 3-phosphate, phosphatidic acid, and lysophosphatidic acid) were also screened. Substitution of these compounds for phosphocholine in the biochemical assay did not yield significant activity. Substitution of deoxyCTP for the CTP in the assay at concentrations up to 10-fold higher also did not yield significant activity.


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Fig. 4.   CCTbeta expression, and effect of CCTbeta overexpression on PtdCho metabolism in COS-7 cells, and lipid stimulation of CCTbeta activity.  A, CCT specific activity in COS-7 cells transfected with plasmids expressing CCTbeta (pPJ34) (bullet ) or an empty vector control (pcDNA3) (open circle ). The cells were harvested and assayed for CCT activity 48 h after transfections with the indicated expression construct as described under "Experimental Procedures." B, effect of CCTbeta overexpression on PtdCho metabolism. COS-7 cells were transfected with the CCTbeta or the control expression vectors, and 36 h later, the cells were labeled with [3H]choline for 24 h. The cells were harvested, and the distribution of label among the soluble choline-derived metabolites and the amounts incorporated into PtdCho were determined by differential extraction and thin-layer chromatography as described under "Experimental Procedures." C, ability of PtdCho:oleic acid to stimulate the activity of CCTbeta . COS-7 cells were transfected with the CCTbeta (pPJ34) expression construct, and 48 h later, the cells were harvested and extracted. Endogenous lipids activators were removed by ion-exchange chromatography, and the ability of added PtdCho:oleic acid (1/1) to stimulate CCTbeta activity was determined as described under "Experimental Procedures." Recovery of CCTbeta activity from the column was >= 90%.

Effect of CCTbeta Expression on PtdCho Metabolism-- COS-7 cells were transfected with the CCTbeta expression plasmid and were labeled with [3H]choline for 24 h to determine whether CCTbeta functions as a CCT in vivo and whether the overexpression of this isozyme effects the PtdCho biosynthetic pathway (Fig. 4B). Although the cellular content of 3H-labeled choline and phosphocholine were the same in control and CCTbeta -transfected cells, there was a 3.4-fold increase in the CDP-choline pool from 1963 ± 13 to 6706 ± 847 cpm/mg. This finding was consistent with the identification of CCTbeta as a phosphocholine cytidylyltransferase and illustrated that overexpression of the enzyme leads to increased accumulation of its product, CDP-choline in vivo. There was also a 50% increase in the incorporation of [3H]choline into PtdCho from 715,228 ± 28,317 to 1.06 × 106 ± 15,959 cpm/mg in CCTbeta -transfected cells indicating that CCTbeta overexpression accelerated PtdCho synthesis. The 5-fold increase in the amount of glycerophosphocholine, a breakdown product of PtdCho, from 7027 ± 251 to 35,835 ± 7083 cpm/mg in the CCTbeta -expressing cells indicated an acceleration of PtdCho turnover similar to that previously observed with CCTalpha overexpression (39).

Lipid Stimulation of CCTbeta Activity-- The similarities in the amphipathic helical domains between CCTalpha and CCTbeta suggested that lipids would stimulate CCTbeta activity in a similar manner to CCTalpha . The addition of the PtdCho:oleic acid lipid activator mixture to crude cell lysates did not enhance CCTbeta activity (not shown). However, the lack of lipid regulation in cell lysates could be attributed to the presence of endogenous lipid activators. This point was tested by removing the endogenous lipid activators from the transfected COS-7 cell lysates by ion-exchange chromatography (16, 23) and determining the ability of PtdCho:oleic acid vesicles to activate the enzyme (Fig. 4C). CCTbeta activity was not detected following the removal of endogenous lipids, and it was potently stimulated by the addition of PtdCho:oleic acid vesicles to the sample. There remain many kinetic details related to the specificity of the lipid regulation of CCTbeta to be investigated with purified CCTbeta , and there may be subtle differences between the two proteins because the amphipathic helical domains are not identical. Nonetheless, our experiments establish that CCTbeta , like CCTalpha , is critically dependent on the presence of stimulatory lipids for activity.

Expression and Modification of CCTbeta Protein-- The predicted molecular size of the CCTbeta protein was confirmed by transcription and translation of the CCTbeta cDNA in vitro using a reticulocyte lysate (Fig. 5). The expressed proteins were radiolabeled with [35S]methionine, and the products were separated by SDS-polyacrylamide gel electrophoresis on 12% gels. A protein of an apparent size of 35 kDa was identified in reactions using CCTbeta cDNA as template and was consistent with the predicted size of 36.3 kDa for CCTbeta protein. As a control, CCTalpha was expressed in the transcription/translation system, and the expected 42-kDa protein was detected (Fig. 5). The CCTbeta expression plasmid was transfected into COS-7 cells, and cell lysates were analyzed for expression of CCTbeta protein by immunoblotting with an antibody raised against amino acids 27-39 of the CCTbeta polypeptide sequence (Fig. 5). Two forms of CCTbeta were detected following expression in COS-7 cells. The less abundant species migrated at the same apparent size as the protein made in vitro (CCTbeta ), and there was a second, slower migrating form (CCTbeta M). The location of both CCTbeta M and CCTalpha at approximately the same position on the gel was not due to cross-reactivity of the two affinity-purified antibodies. The specificities of the anti-CCTalpha and anti-CCTbeta amino-terminal antibodies were clearly demonstrated in the same experiment (Fig. 5). The larger apparent size of CCTbeta M suggests that a significant portion of the expressed protein is modified posttranslationally.


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Fig. 5.   Antibody specificity and in vivo modification of CCTbeta . Left three lanes, transcription and translation in vitro of CCTalpha or CCTbeta cDNAs using a reticulocyte lysate system (Promega) containing 40 µCi/50 µl [35S]methionine (1000 Ci/mmol). The products were separated by SDS gel electrophoresis on 12% gels, and the bands were visualized by autoradiography. Middle and right lanes, cellular expression of CCTalpha or CCTbeta protein species. COS-7 cells were transfected with pcDNA3 plasmids carrying CCTalpha or CCTbeta cDNAs, and cell lysates were analyzed by immunoblotting 48 h later. Samples (50 µg protein) of the total cell lysates were probed with either anti-CCTalpha or anti-CCTbeta antibodies 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.

A second approach was used to verify that a posttranslational modification of the CCTbeta sequence was responsible for the retarded electrophoretic mobility of CCTbeta M. The cDNA sequences corresponding to the dissimilar amino-terminal regions of CCTalpha and CCTbeta were exchanged. Proteins were expressed from the chimeric cDNAs that represented the first 84 amino acids of CCTbeta followed by the remaining 283 amino acids of CCTalpha (CCTbeta /CCTalpha , Fig. 6) or the first 84 amino acids of CCTalpha followed by the latter 246 amino acids of CCTbeta (CCTalpha /CCTbeta , Fig. 6). The anti-CCTbeta antibody recognized both the authentic CCTbeta and CCTbeta M proteins (Fig. 6, lane 8) expressed in COS-7 cells as well as the CCTbeta /CCTalpha and CCTbeta M/CCTalpha chimeras (lane 6). The CCTbeta /CCTalpha chimera exhibited a larger apparent molecular size than CCTbeta as anticipated due to the longer carboxyl terminus of CCTalpha (lane 6) and also displayed the previously reported gel shifts due to multiple phosphorylation of the CCTalpha carboxyl-terminal domain (40). In contrast, the anti-CCTalpha antibody only detected the gel shifts due to phosphorylation of the authentic CCTalpha protein (lane 4) and did not reveal any modification of the smaller CCTalpha /CCTbeta chimeric protein (lane 7). The dissimilarities of the CCTalpha and CCTbeta NH2 termini, the demonstrated specificities of the antibodies, and the reproduction of the gel shift with in vivo expression of a CCTbeta /CCTalpha fusion protein strongly support the idea that CCTbeta is biochemically modified. This modification results in a protein that migrates with approximately the same apparent molecular weight as CCTalpha following denaturing gel electrophoresis.


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Fig. 6.   CCTbeta amino terminus specifies posttranslational modification. COS-7 cells were transfected with pcDNA3 vector alone or pcDNA3 vectors carrying cDNAs encoding CCTalpha , CCTbeta , CCTalpha /CCTbeta chimeric protein, CCTbeta /CCTalpha chimeric protein, or CCTbeta [Delta 1-26]. Cell lysates were prepared after 48 h, samples containing 50 µg of protein were separated on 12% SDS gels, and expressed proteins were immunoblotted with either anti-CCTalpha or anti-CCTbeta affinity-purified antibodies as described under "Experimental Procedures."

To further localize the sequences in CCTbeta required for the posttranslational modification, the first 26 amino acids were deleted from the NH2 terminus. The truncated protein, called CCTbeta [Delta 1-26], was expressed in COS-7 cells and detected by immunoblotting with the anti-CCTbeta antibody (Fig. 6). The apparent molecular size of CCTbeta [Delta 1-26] was smaller than CCTbeta , as expected; however, a CCTbeta M[Delta 1-26] species was not observed, indicating that posttranslational modification of the truncated protein did not occur (Fig. 6, lane 11). These data suggested that the first 26 amino acids of CCTbeta were necessary for cellular processing of the protein.

Regulation of CCTbeta Activity-- CCTbeta activity was examined in vitro to determine whether the NH2-terminal sequence and modification of the protein influenced expression and/or catalytic activity. COS-7 cells were transfected with the plasmids encoding CCTbeta , CCTalpha , the chimeric CCT proteins (CCTbeta /CCTalpha and CCTalpha /CCTbeta ), and the NH2-terminal truncated CCTbeta [Delta 1-26]. The CCT protein species were expressed at approximately equivalent levels based on the immunoblots (Fig. 6). The CCT protein species in the cell lysates were assayed in the presence of excess stimulatory lipids to evaluate their relative activities. Although cells overexpressing (CCTbeta plus CCTbeta M) had higher activity (76 nmol/min/mg) than the endogenous control activity (8 nmol/min/mg), [CCTbeta plus CCTbeta M] was less active than CCTalpha and its multiply phosphorylated species (1105 nmol/min/mg) (Fig. 7). Substitution of the NH2-terminal domain of CCTalpha onto CCTbeta enhanced biochemical activity (215 nmol/min/mg), whereas the amino-terminal domain of CCTbeta dramatically reduced the activity of CCTalpha (91 nmol/min/mg). These data suggested that the protein modification directed by the amino terminus of CCTbeta attenuates biochemical activity. In support of this hypothesis, truncation of the first 26 amino acids of CCTbeta elevated activity almost 10-fold (686 nmol/min/mg), supporting the idea that the NH2 terminus plays a role in the cellular regulation of CCTbeta activity.


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Fig. 7.   The CCTbeta amino terminus inhibits catalytic activity. COS-7 cells were transfected with pcDNA3 constructs encoding CCTalpha , CCTbeta , CCTalpha /CCTbeta chimeric protein, CCTbeta /CCTalpha chimeric protein, or CCTbeta [Delta 1-26]. After 48 h, cell lysates (10 µg of protein) were assayed for CCT biochemical activity as described under "Experimental Procedures." Control lysates were obtained from cells transfected with pcDNA3 vector alone.

Cellular Localization of CCTbeta -- The amino terminus of CCTbeta bears little resemblance to the amino terminus of CCTalpha , which harbors a nuclear localization sequence. CCTalpha is reported to be predominately an intranuclear protein based on indirect immunofluorescence using an antibody raised against an amino-terminal peptide of CCTalpha (7, 8). The lack of a nuclear localization motif in CCTbeta suggested that it would not be found in the cell nucleus. This hypothesis was tested by evaluating the distribution of CCTbeta in human HeLa cells using the anti-CCTbeta peptide antibody and indirect immunofluorescence microscopy (Fig. 8). The affinity-purified peptide antibody detected CCTbeta protein in the cytoplasm and did not detect the protein in the nucleus, even at the lowest antibody dilutions. The cytoplasmic staining appeared diffuse but was higher than the apparent background staining of the nucleus. The background signal associated with the cell nucleus/nucleolus was due to reaction with the fluorescein-conjugated secondary antibodies used in the assay (Fig. 8B). Monoclonal anti-p120 antibody positively identified the nucleolus in these cells (Fig. 8C), and anti-vimentin monoclonal antibody, which signaled the cytoskeleton, was used as a cytoplasmic marker (Fig. 8D). These data confirm that CCTbeta is localized primarily outside of the nucleus.


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Fig. 8.   Immunofluorescence micrographs of HeLa cells labeled with anti-CCTbeta antibodies. A, cells were incubated with anti-CCTbeta as the primary antibody. B, cells were labeled with fluorescein-conjugated secondary antibodies only. C, cells were labeled with anti-p120 antibodies as a marker for the nucleolus. D, cells were labeled with anti-vimentin antibodies to visualize the cytoskeleton in the cytoplasm. N designates the nucleus, and Nu designates the nucleolus.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The existence of a second isoform of CCT opens the door to a series of experiments to determine the physiological function of CCTbeta . Our data indicate that the biochemical properties of the two CCT isoforms are similar and the overexpression of either isoform is capable of perturbing PtdCho biosynthesis and metabolism. However, the distinct differences between the amino and carboxyl-terminal domains of CCTalpha and CCTbeta indicate that the two isoforms likely have unique regulatory properties. The two isoforms are clearly the products of different genes and are differentially expressed in tissues and perhaps also during development. Because CCTalpha is the only isoform detected in the large volume of work in this area (1, 2), it is possible that CCTbeta is expressed at lower levels or in a specific developmental settings compared with than the more widely distributed CCTalpha .

Previous data may have to be reinterpreted in light of the existence of CCTbeta . For example, data on whole tissue CCT activity and distribution will need to be reevaluated. The Northern blots cannot be used to predict the relative levels of CCTalpha and CCTbeta proteins in particular tissues. Total CCT specific activity is relatively low in most tissues, illustrating that neither isoform is expressed at high levels. The properties of CCTbeta when expressed in vivo and assayed in vitro suggest that there are no significant differences in biochemical characteristics that could distinguish between the two isozymes in crude extracts. The general impression is that CCTbeta may be more restrictive in its expression compared with CCTalpha because CCTalpha has been the only mammalian isoform cloned to date. Nonetheless, we think that these data will be important to gather because some of the controversial issues in the CCT field may be explained by the presence of two isoforms.

The CCTalpha anti-peptide antibody used previously to identify its nuclear localization (7, 8) does not cross-react with CCTbeta (Figs. 5 and 6). Houweling et al. (24) report that CCT is both a nuclear and cytoplasmic protein in primary hepatocytes using a peptide antibody raised against amino acids 164-176 of CCTalpha . This sequence in the 164-176 region (DFVAHDDIPYSSA) is identical in human CCTalpha and CCTbeta ; therefore, antisera raised against this peptide would be predicted to react with both CCT isoforms. We detect both CCTalpha and CCTbeta transcripts in rodent liver,2 and it will be very interesting to determine whether the cytoplasmic CCT detected in primary rodent hepatocytes can be attributed to the presence of CCTbeta in this tissue.

The co-migration of CCTalpha and CCTbeta M on denaturing gels and their similar reliance on lipid activators for biochemical activity makes it difficult to determine whether CCTbeta is a component of purified CCT preparations from mammalian sources. The regulation of CCTalpha is governed by phosphorylation of its carboxyl terminus at multiple sites, resulting in at least two species that migrate more slowly on denaturing gels (Figs. 5 and 6 and Ref. 40). Phosphorylation interferes with the stimulatory action of lipids on CCTalpha activity (23), and there is some correlation with membrane dissociation of CCTalpha in vivo (20), but the regulation by this mechanism is not an absolute on/off switch (23, 35). On the other hand, the activity of CCTbeta M, a protein with approximately the same molecular weight as CCTalpha , is lower (Fig. 7) due to posttranslational modification that is dependent on the amino terminus. The nature of the modification of CCTbeta protein and its role in the regulation of PtdCho biosynthesis is currently under investigation.

    ACKNOWLEDGEMENTS

We thank Pam Jackson for her expert technical assistance, Wannian Yang for the construction of pWYCT, and Chuck Rock for his comments on the research. We thank Human Genome Sciences for providing us with information on the CCT-like cDNA contained in their library.

    FOOTNOTES

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

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF052510.

** 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{at}stjude.org.

1 The abbreviations used are: CCT, CTP:phosphocholine cytidylyltransferase; PtdCho, phosphatidylcholine; alpha -isoform, the previously discovered CCT; beta -isoform, the new CCT described in this paper; kb, kilobase(s); bp, base pair(s).

2 A. Lykidis and S. Jackowski, unpublished observations.

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Abstract
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Procedures
Results
Discussion
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