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• W2050477788 abstract "CTPphosphocholine cytidylyltransferase is a major regulator of phosphatidylcholine biosynthesis. A single isoform, CCTα, has been studied extensively and a second isoform, CCTβ, was recently identified. We identify and characterize a third cDNA, CCTβ2, that differs from CCTβ1 at the carboxyl-terminal end and is predicted to arise as a splice variant of the CCTβ gene. Like CCTα, CCTβ2 is heavily phosphorylated in vivo, in contrast to CCTβ1. CCTβ1 and CCTβ2 mRNAs were differentially expressed by the human tissues examined, whereas CCTα was more uniformly represented. Using isoform-specific antibodies, both CCTβ1 and CCTβ2 localized to the endoplasmic reticulum of cells, in contrast to CCTα which resided in the nucleus in addition to associating with the endoplasmic reticulum. CCTβ2 protein has enzymatic activity in vitro and was able to complement the temperature-sensitive cytidylyltransferase defect in CHO58 cells, just as CCTα and CCTβ1 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 CCTβ1 and CCTβ2 may play a role that is distinct from ubiquitously expressed CCTα. phosphocholine cytidylyltransferase is a major regulator of phosphatidylcholine biosynthesis. A single isoform, CCTα, has been studied extensively and a second isoform, CCTβ, was recently identified. We identify and characterize a third cDNA, CCTβ2, that differs from CCTβ1 at the carboxyl-terminal end and is predicted to arise as a splice variant of the CCTβ gene. Like CCTα, CCTβ2 is heavily phosphorylated in vivo, in contrast to CCTβ1. CCTβ1 and CCTβ2 mRNAs were differentially expressed by the human tissues examined, whereas CCTα was more uniformly represented. Using isoform-specific antibodies, both CCTβ1 and CCTβ2 localized to the endoplasmic reticulum of cells, in contrast to CCTα which resided in the nucleus in addition to associating with the endoplasmic reticulum. CCTβ2 protein has enzymatic activity in vitro and was able to complement the temperature-sensitive cytidylyltransferase defect in CHO58 cells, just as CCTα and CCTβ1 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 CCTβ1 and CCTβ2 may play a role that is distinct from ubiquitously expressed CCTα. phosphatidylcholine CTP:phosphochoine cytidylyltransferase cytidine diphosphocholine reverse transcription-polymerase chain reaction base pair phosphate-buffered saline. CHO, Chinese hamster ovary endoplasmic reticulum 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)- propane-1,3-diol polyacrylamide gel electrophoresis 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 (1Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar) and membrane-protein interaction is one important mechanism that governs cellular CCT activity (1Kent C. Biochim. Biophys. Acta. 1997; 1348: 79-90Crossref PubMed Scopus (192) Google Scholar, 2Cornell R.B. Gross R.W. Advances in Lipobiology. JAI Press, Greenwich, CT1996: 1-38Google Scholar). Recently a second isoform, CCTβ, was discovered which is encoded by a second gene (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). CCTα and CCTβ 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 (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 4Cornell R.B. Biochemistry. 1991; 30: 5881-5888Crossref PubMed Scopus (74) Google Scholar, 5Luche M.M. Rock C.O. Jackowski S. Arch. Biochem. Biophys. 1993; 301: 114-118Crossref PubMed Scopus (26) Google Scholar, 6Boggs K.P. Rock C.O. Jackowski S. J. Biol. Chem. 1995; 270: 7757-7764Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, 7Wang Y. Kent C. J. Biol. Chem. 1995; 270: 18948-18952Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 8Yang W. Boggs K.P. Jackowski S. J. Biol. Chem. 1995; 270: 23951-23957Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Shanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar), 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 α-helices upon association with phospholipid regulators (10Craig L. Johnson J.E. Cornell R.B. J. Biol. Chem. 1994; 269: 3311-3317Abstract Full Text PDF PubMed Google Scholar, 11Johnson J.E. Cornell R.B. Biochem. J. 1994; 33: 4327-4335Crossref Scopus (67) Google Scholar, 12Dunne S.J. Cornell R.B. Johnson J.E. Glover N.R. Tracey A.S. Biochemistry. 1996; 35: 11975-11984Crossref PubMed Scopus (91) Google Scholar, 13Johnson J.E. Aebersold R. Cornell R.B. Biochim. Biophys. Acta. 1997; 1324: 273-284Crossref PubMed Scopus (39) Google Scholar). The amino terminus of CCTβ bears no resemblance to the amino terminus of CCTα and does not include a nuclear localization sequence as was identified in the CCTα protein (14Yamagishi M. Matsushima H. Wada A. Sakagami M. Fujita N. Ishihama A. EMBO J. 1993; 12: 625-630Crossref PubMed Scopus (123) Google Scholar, 15Wang Y. MacDonald J.I.S. Kent C. J. Biol. Chem. 1995; 270: 354-360Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). CCTα has been localized predominantly in the nucleus but the physiological significance of the nuclear localization of CCTα remains unclear. CCTβ protein was localized outside the cell nucleus by indirect immunofluorescent microscopy (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). CCTβ consists of 330 amino acids, in contrast with the 367 residues of CCTα, and lacks most of the carboxyl-terminal phosphorylation domain that is found in the CCTα protein (9Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Shanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar, 16MacDonald J.I.S. Kent C. J. Biol. Chem. 1994; 269: 10529-10537Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of CCTα interferes with the lipid stimulation of enzyme activity in vitro (17Yang W. Jackowski S. J. Biol. Chem. 1995; 270: 16503-16506Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) and correlates with a reduction of PtdCho biosynthesis in vivo (18Pelech S.L. Vance D.E. J. Biol. Chem. 1982; 257: 14198-14202Abstract Full Text PDF PubMed Google Scholar, 19Watkins J.D. Wang Y. Kent C. Arch. Biochem. Biophys. 1992; 292: 360-367Crossref PubMed Scopus (23) Google Scholar, 20Watkins J.D. Kent C. J. Biol. Chem. 1990; 265: 2190-2197Abstract Full Text PDF PubMed Google Scholar, 21Watkins J.D. Kent C. J. Biol. Chem. 1991; 266: 21113-21117Abstract Full Text PDF PubMed Google Scholar, 22Wang Y. MacDonald J.I.S. Kent C. J. Biol. Chem. 1993; 268: 5512-5518Abstract Full Text PDF PubMed Google Scholar, 23Houweling M. Jamil H. Hatch G.M. Vance D.E. J. Biol. Chem. 1994; 269: 7544-7551Abstract Full Text PDF PubMed Google Scholar, 24Wang Y. Kent C. J. Biol. Chem. 1995; 270: 17843-17849Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Despite the differences at the amino and carboxyl termini of the proteins, both CCTα and CCTβ exhibit high activity when overexpressed in COS-7 cells (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 9Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Shanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar, 25Johnson J.E. Kalmar G.B. Sohal P.S. Walkey C.J. Yamashita S. Cornell R.B. Biochem. J. 1992; 285: 815-820Crossref PubMed Scopus (45) Google Scholar, 26Walkey C.J. Kalmar G.B. Cornell R.B. J. Biol. Chem. 1994; 269: 5742-5749Abstract Full Text PDF PubMed Google Scholar) resulting in accumulation of cellular CDP-choline and increased radiolabeling of PtdCho (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 27Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 9400-9408Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In this work we identify a third isoform of CCT, called CCTβ2, which is a splice variant of CCTβ. CCTβ2 encodes a 369-amino acid protein which is identical to the CCTβ1 isoform described previously from amino acids 1 to 320. However, CCTβ2 also has a carboxyl-terminal sequence that resembles the phosphorylation domain of CCTα. The existence of two distinct CCT genes and two CCTβ splice variants raises the possibility of regulation of CCT activity at the level of gene expression as well as subcellular localization and phosphorylation (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Thus, we investigated the expression of the CCT isoforms in human tissues, determine whether CCTβ2 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 (28Esko J.D. Wermuth M.M. Raetz C.R.H. J. Biol. Chem. 1981; 256: 7388-7393Abstract Full Text PDF PubMed Google Scholar). 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 GreenTM 488; Texas RedTM, Hoechst and FluoReporterTM labeling kits, Oregon GreenTM, and Texas RedTM, concanavalin A, and wheat germ agglutinin conjugates, ProlongTM antifade kit with mounting medium; Nalge Nunc International, LabTekTM II Chamber SlidesTM; 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 cloneAI041180; Sigma, anti-FLAG M2 monoclonal antibody, CTP, phosphocholine and buffers; Analtech, thin-layer chromatography plates. All other supplies were reagent grade or better. Anti-CCTα rabbit polyclonal antiserum was raised against a synthetic peptide (MDAQSSAKVNSRKRRKE) corresponding to the first 17 amino acids of CCTα. Anti-CCTβ antibody (B1 epitope) was a rabbit polyclonal antiserum raised against a peptide (MEEIEHTCPQPRL) corresponding to amino acids 27–39 of CCTβ1 and CCTβ2. The anti-CCTβ antibody (B2 epitope) was a rabbit polyclonal antiserum raised against a synthetic peptide (TTDAESETGIPKSLSNEP) corresponding to amino acids 5–22 of CCTβ1 and CCTβ2. Anti-CCTβ2 antibody (B3 epitope) was a rabbit polyclonal antiserum raised against a synthetic peptide (PPSSPKAASRSISSMSEGD) corresponding to amino acids 347–365 of CCTβ2. Resequencing of the CCTβ2 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 CCTβ1 and CCTβ2 whereas the B3 epitope antibody recognized only CCTβ2. 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 (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The EST data base was searched using the published CCTβ sequence (GenBankTM/EBI Data Bank accession numberAF052510). A clone from human brain was identified (GenBankTM 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 (GenBankTM 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 Bam HI and Xho I (pAL1). pcDNA3 has an Ssp I site approximately 1 kilobase from the 5′ end of the T7 promoter and pAL1 retains the Ssp I site of CCTβ. The cDNA encoding CCTβ1 in pcDNA3 (pPJ34) (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) was also digested with Ssp I. The approximately 1.1-kilobase fragment derived from plasmid pPJ34 was ligated to the 5.5-kilobase fragment of pAL1 to generate pAL2. The M27A point mutation was constructed using overlap extension PCR with the CCTβ1 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 Sna B1 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-Sna B1rev 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 Sna B1rev. The 642-bp product was cloned into pCR2.1 (plasmid pPJ76) and sequenced to verify that it had the desired mutation. The Bam HI-Sna BI fragment of pPJ76 was ligated into the CCTβ1 cDNA replacing the Bam HI-Sna BI fragment of AA382871. The mutated CCTβ1 cDNA was subcloned into pcDNA3 using Bam HI-Xho I. CCT activity was determined essentially as described previously (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The standard assay contained 150 mm bis-Tris-HCl, pH 6.5, 10 mmMgCl2, 4 mm CTP, 64 μm lipid activator (PtdCho:oleic acid, 1:1), 1 mmphospho[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 0.5m 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 (29Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar). 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. COS-7 cells were grown in 100-mm dishes and transfected with 10 μg of vector expressing CCTβ1, CCTβ2, 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). HeLa cell cultures were harvested and total RNA was isolated by a guanidine isothiocyanate lysis procedure followed by pelleting RNA by CsCl gradient centrifugation (30Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1977; 18: 5294-5299Crossref Scopus (16652) Google Scholar). RNA pellets dissolved in 10 mm Tris-HCl, pH 7.5, 5% β-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 (31Kingston R.E. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Current Protocols in Molecular Biology. John Wiley & Sons, Inc., New York1994: 4.5.1-4.5.3Google Scholar). 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 CCTα expression was 5′-GAAGGTGGAGGAAAAAAG-3′ corresponding to 795–812 bp of the CCTα cDNA sequence, and the reverse primer was 5′-ACAGAAAGGGAGGACAG-3′ corresponding to 1123–1159 bp of the CCTα cDNA sequence. The forward primer for CCTβ was 5′-CAAGTGGACAAAATGAAGG-3′ corresponding to 733–751 bp of the CCTβ cDNA sequence and the reverse primer was 5′-CTAGAAGTCTCTGCACCTCG-3′ corresponding to 1299–1238 bp of the CCTβ2 sequence or 974–993 bp of the CCTβ1 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. 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. 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-CCTα (1:2000 dilution), purified anti-CCTβ1 (B2 epitope) (1:2000 dilution), or purified anti-CCTβ2 (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 mmNaCl, 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-CCTβ (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. The antibodies were labeled according to the instructions provided with Molecular Probes' FluoReporterTM labeling kits. Briefly, 200 μl of the 1–2 mg/ml antibody in PBS was combined with 20 ml of 1m 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 × MWreactive dye × 200 ×MR)/MWprotein. 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. BAC1.2F5 cells (32Jackowski S. J. Biol. Chem. 1994; 269: 3858-3867Abstract Full Text PDF PubMed Google Scholar) and HeLa cells (33Baburina I. Jackowski S. J. Biol. Chem. 1998; 273: 2169-2173Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar) were cultured as described previously. Cells were grown in 4-chamber LabTekTM II Chamber Slides™. 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 GreenTM-labeled anti-CCTα 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 (15Wang Y. MacDonald J.I.S. Kent C. J. Biol. Chem. 1995; 270: 354-360Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar); 2) fixation in 3.7% formaldehyde for 20 min and permeabilization with cold acetone for 20 min at −20 °C (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar); 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 mmglycine for 15 min at −20 °C. All of the procedures, except the last one, resulted in the distribution of anti-CCTα staining in both nuclear and cytoplasmic compartments. Using the last fixation/permeabilization procedure, anti-CCTα 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 ProlongTM 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 GreenTM 488 fluorophore was visualized using an argon-ion laser and a fluorescein isothiocyanate filter set (488, 514 nm); Texas RedTM 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. Two human cDNA clones similar to CCTβ1 were identified (GenBankTM 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 CCTβ mRNA, called CCTβ2, that was identical to CCTβ1 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 CCTβ 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 β2 carboxyl terminus precedes the one encoding the β1 terminus in the genomic structure. The sequence analysis indicated that CCTβ2 is a splice variant of CCTβ1. The cDNA for CCTβ2 was assembled to exclude the possibility of expression of CCTβ1, and subcloned into the expression vector pcDNA3. The predicted amino acid sequence of CCTβ2 was aligned with the sequences of CCTα and CCTβ1 (Fig. 2). The predicted CCTβ2 protein had 369 amino acids and was identical to CCTβ1 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 CCTα and missing from CCTβ1. The carboxyl terminus of CCTβ2 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 CCTβ2 align with the corresponding residues of CCTα. The existence of 21 potential serine and threonine phosphorylation sites in the predicted carboxyl-terminal domain of CCTβ2 suggested that this enzyme was phosphorylated similar to the modification of CCTα protein. This point was tested by transfecting COS-7 cells with CCTβ1, CCTβ2, or a vector control and followed labeling 48 h later with [32P]orthophosphate (160 μCi/ml) for 60 min. Both CCTβ isoforms were immunoprecipitated with the amino-terminal anti-CCTβ antibody (B2 epitope), fractionated by SDS-PAGE, and the radiolabeled proteins were visualized by autoradiography. CCTβ2 was highly phosphorylated (Fig. 3) confirming the prediction made from the analysis of the primary structure of its carboxyl terminus. CCTβ1 was also phosphorylated, although to a signficantly lesser extent as was predicted from the fact that CCTβ1 had only 3 potential phosphorylation sites after amino acid 310. These data are consistent with the idea that the carboxyl-terminal domains of CCTβ1 and CCTβ2 were the exclusive sites of phosphorylation, as was shown with CCTα (9Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Shanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar, 16MacDonald J.I.S. Kent C. J. Biol. Chem. 1994; 269: 10529-10537Abstract Full Text PDF PubMed Google Scholar). In our previous report (3Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114" @default.
• W2050477788 created "2016-06-24" @default.
• W2050477788 creator A5055111001 @default.
• W2050477788 creator A5085170224 @default.
• W2050477788 creator A5090935512 @default.
• W2050477788 date "1999-09-01" @default.
• W2050477788 modified "2023-10-12" @default.
• W2050477788 title "Distribution of CTP:Phosphocholine Cytidylyltransferase (CCT) Isoforms" @default.
• W2050477788 cites W135018339 @default.
• W2050477788 cites W1487624404 @default.
• W2050477788 cites W1490431590 @default.
• W2050477788 cites W1514632623 @default.
• W2050477788 cites W1527594904 @default.
• W2050477788 cites W1538432305 @default.
• W2050477788 cites W1545450687 @default.
• W2050477788 cites W1565685625 @default.
• W2050477788 cites W1592254226 @default.
• W2050477788 cites W1601458675 @default.
• W2050477788 cites W1647092045 @default.
• W2050477788 cites W18820718 @default.
• W2050477788 cites W1909266718 @default.
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• W2050477788 cites W2043988433 @default.
• W2050477788 cites W2048530944 @default.
• W2050477788 cites W2050532520 @default.
• W2050477788 cites W2056261849 @default.
• W2050477788 cites W2059606380 @default.
• W2050477788 cites W2060333964 @default.
• W2050477788 cites W2063032509 @default.
• W2050477788 cites W2064859908 @default.
• W2050477788 cites W2067052397 @default.
• W2050477788 cites W2073810769 @default.
• W2050477788 cites W2088884715 @default.
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