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• W2014089999 abstract "Enzyme deficiency in the salvage pathway of deoxyribonucleotide synthesis in mitochondria can cause mtDNA depletion syndromes. We have identified a human mitochondrial UMP-CMP kinase (UMP-CMPK, cytidylate kinase; EC 2.7.4.14), designated as UMP-CMP kinase 2 (UMP-CMPK2). The C-terminal domain of this 449-amino acid protein contains all consensus motifs of a nucleoside monophosphate kinase. Phylogenetic analysis showed that UMP-CMPK2 belonged to a novel nucleoside monophosphate kinase family, which was closer to thymidylate kinase than to cytosolic UMP-CMP kinase. Subcellular localization with green fluorescent protein fusion proteins illustrated that UMP-CMPK2 was localized in the mitochondria of HeLa cells and that the mitochondrial targeting signal was included in the N-terminal 22 amino acids. The enzyme was able to phosphorylate dUMP, dCMP, CMP, and UMP with ATP as phosphate donor, but the kinetic properties were different compared with the cytosolic UMP-CMPK. Its efficacy to convert dUMP was highest, followed by dCMP, whereas CMP and UMP were the poorest substrates. It also phosphorylated the monophosphate forms of the nucleoside analogs ddC, dFdC, araC, BVDU, and FdUrd, which suggests that UMP-CMPK2 may be involved in mtDNA depletion caused by long term treatment with ddC or other pyrimidine analogs. UMP-CMPK2 mRNA expression was exclusively detected in chronic myelogenous leukemia K-562 and lymphoblastic leukemia MOLT-4 among eight studied cancer cell lines. Particular high expression in leukemia cells, dominant expression in bone marrow, and tight correlation with macrophage activation and inflammatory response suggest that UMP-CMPK2 may have other functions in addition to the supply of substrates for mtDNA synthesis. Enzyme deficiency in the salvage pathway of deoxyribonucleotide synthesis in mitochondria can cause mtDNA depletion syndromes. We have identified a human mitochondrial UMP-CMP kinase (UMP-CMPK, cytidylate kinase; EC 2.7.4.14), designated as UMP-CMP kinase 2 (UMP-CMPK2). The C-terminal domain of this 449-amino acid protein contains all consensus motifs of a nucleoside monophosphate kinase. Phylogenetic analysis showed that UMP-CMPK2 belonged to a novel nucleoside monophosphate kinase family, which was closer to thymidylate kinase than to cytosolic UMP-CMP kinase. Subcellular localization with green fluorescent protein fusion proteins illustrated that UMP-CMPK2 was localized in the mitochondria of HeLa cells and that the mitochondrial targeting signal was included in the N-terminal 22 amino acids. The enzyme was able to phosphorylate dUMP, dCMP, CMP, and UMP with ATP as phosphate donor, but the kinetic properties were different compared with the cytosolic UMP-CMPK. Its efficacy to convert dUMP was highest, followed by dCMP, whereas CMP and UMP were the poorest substrates. It also phosphorylated the monophosphate forms of the nucleoside analogs ddC, dFdC, araC, BVDU, and FdUrd, which suggests that UMP-CMPK2 may be involved in mtDNA depletion caused by long term treatment with ddC or other pyrimidine analogs. UMP-CMPK2 mRNA expression was exclusively detected in chronic myelogenous leukemia K-562 and lymphoblastic leukemia MOLT-4 among eight studied cancer cell lines. Particular high expression in leukemia cells, dominant expression in bone marrow, and tight correlation with macrophage activation and inflammatory response suggest that UMP-CMPK2 may have other functions in addition to the supply of substrates for mtDNA synthesis. Nucleotide synthesis is a basic biological process for cell proliferation and almost all other physiological activities in the cell. Two pathways have been reported for nucleotide synthesis: the de novo pathway and the salvage pathway (1Van Rompay A.R. Johansson M. Karlsson A. Pharmacol. Therap. 2000; 87: 189-198Crossref PubMed Scopus (178) Google Scholar). In the de novo pathway, the synthesis of nucleotides starts from small molecules whereas in the salvage pathway, free nucleosides are directly used to synthesize ribonucleotides and deoxyribonucleotides. Mitochondria only have the salvage pathway for nucleotide synthesis. To our knowledge, there are seven enzymes of this pathway that have been cloned and studied in human tissues: thymidine kinase 2 (TK2) 2The abbreviations used are: TK2thymidine kinase 2DTTdithiothreitolGFPgreen fluorescent proteinmtDNAmitochondrial DNAddC2′,3′-dideoxycytidinedFdC2′,2′-difluorodeoxycytidinearaC1-β-d-arabinofuranosylcytosineBVDU(E)-5-(2-bromovinyl)-2′-deoxyuridineBVaraU1-β-d-arabinofuranosyl-5-(E)-(2-bromovinyl)uracilFdUrd5-fluorodeoxyuridineddIdideoxyinosineLPSlipopolysaccharideTNFαtumor necrosis factor αIFNαα-interferonIFNγγ-interferonIL-1βinterleukin-1βTGFβtransforming growth factor βUMP-CMPKUMP-CMP kinase. (2Munch-Petersen B. Cloos L. Tyrsted G. Eriksson S. J. Biol. Chem. 1991; 266: 9032-9038Abstract Full Text PDF PubMed Google Scholar, 3Johansson M. Karlsson A. J. Biol. Chem. 1997; 272: 8454-8458Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 4Wang L. Saada A. Eriksson S. J. Biol. 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Chem. 2003; 278: 46195-46198Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), deoxyguanosine kinase (dGK) (10Johansson M. Bajalica-Lagercrantz S. Lagercrantz J. Karlsson A. Genomics. 1996; 38: 450-451Crossref PubMed Scopus (15) Google Scholar, 11Johansson M. Karlsson A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7258-7262Crossref PubMed Scopus (81) Google Scholar, 12Wang L. Hellman U. Eriksson S. FEBS Letts. 1996; 390: 39-43Crossref PubMed Scopus (55) Google Scholar, 13Zhu C. Johansson M. Permert J. Karlsson A. J. Biol. Chem. 1998; 273: 14707-14711Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), adenylate kinase 2 (AK2) (14Noma T. Song S. Yoon Y.-S. Tanaka S. Nakazawa A. Biochim. Biophys. Acta, Gene Structure and Expression. 1998; 1395: 34-39Crossref PubMed Scopus (29) Google Scholar), adenylate kinase 3 (AK3) (15Noma T. Fujisawa K. Yamashiro Y. Shinohara M. Nakazawa A. Gondo T. Ishihara T. Yoshinobu K. Biochem. J. 2001; 358: 225-232Crossref PubMed Scopus (64) Google Scholar), adenylate kinase 3-like 1 (AK3L1, also known as AK4) (15Noma T. Fujisawa K. Yamashiro Y. Shinohara M. Nakazawa A. Gondo T. Ishihara T. Yoshinobu K. Biochem. J. 2001; 358: 225-232Crossref PubMed Scopus (64) Google Scholar, 16Yoneda T. Sato M. Maeda M. Takagi H. Mol. Brain Res. 1998; 62: 187-195Crossref PubMed Scopus (53) Google Scholar), and nucleoside diphosphate kinase NME4 (nm23-H4) (17Milon L. Rousseau-Merck M.-F. Munier A. Erent M. Lascu I. Capeau J. Lacombe M.L. Hum. Genet. 1997; 99: 550-557Crossref PubMed Scopus (112) Google Scholar, 18Milon L. Meyer P. Chiadmi M. Munier A. Johansson M. Karlsson A. Lascu I. Capeau J. Janin J. Lacombe M.-L. J. Biol. Chem. 2000; 275: 14264-14272Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Although the Drosophila melanogaster UMP-CMP kinase was reported to be a mitochondrial enzyme (19Curbo S. Amiri M. Foroogh F. Johansson M. Karlsson A. Biochem. Biophys. Res. Commun. 2003; 311: 440-445Crossref PubMed Scopus (10) Google Scholar), there are no reports on a human mitochondrial UMP-CMP kinase or thymidylate kinase so far. thymidine kinase 2 dithiothreitol green fluorescent protein mitochondrial DNA 2′,3′-dideoxycytidine 2′,2′-difluorodeoxycytidine 1-β-d-arabinofuranosylcytosine (E)-5-(2-bromovinyl)-2′-deoxyuridine 1-β-d-arabinofuranosyl-5-(E)-(2-bromovinyl)uracil 5-fluorodeoxyuridine dideoxyinosine lipopolysaccharide tumor necrosis factor α α-interferon γ-interferon interleukin-1β transforming growth factor β UMP-CMP kinase. Antiretroviral or anticancer deoxynucleoside analogs can cause mtDNA depletion and lead to mitochondria dysfunction after long term treatment (20Pan-Zhou X.-R. Cui L. Zhou X.-J. Sommadossi J.-P. Darley-Usmar V.M. Antimicrob. Agents Chemother. 2000; 44: 496-503Crossref PubMed Scopus (119) Google Scholar, 21Cote H.C.F. Brumme Z.L. Craib K.J.P. Alexander C.S. Wynhoven B. Ting L. Wong H. Harris M. Harrigan P.R. O'Shaughnessy M.V. Montaner J.S.G. N. Engl. J. Med. 2002; 346: 811-820Crossref PubMed Scopus (516) Google Scholar). Mutations or deletion of either TK2 or dGK result in myopathic or hepatocerebral forms of mtDNA depletion syndromes (MDS) (22Saada A. Shaag A. Mandel H. Nevo Y. Eriksson S. Elpeleg O. Nat. Genet. 2001; 29: 342-344Crossref PubMed Scopus (496) Google Scholar, 23Tulinius M. Moslemi A.-R. Darin N. Holme E. Oldfors A. Neuromusc. Disorders. 2005; 15: 412-415Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 24Salviati L. Sacconi S. Mancuso M. Otaegui D. Camano P. Marina A. Rabinowitz S. Shiffman R. Thompson K. Wilson C.M. Feigenbaum A. Naini A.B. Hirano M. Bonilla E. DiMauro S. Vu T.H. Ann. Neurol. 2002; 52: 311-317Crossref PubMed Scopus (142) Google Scholar, 25Wang L. Limongelli A. Vila M.R. Carrara F. Zeviani M. Eriksson S. Mol. Genet. Metab. 2005; 84: 75-82Crossref PubMed Scopus (65) Google Scholar, 26Freisinger P. Futterer N. Lankes E. Gempel K. Berger T.M. Spalinger J. Hoerbe A. Schwantes C. Lindner M. Santer R. Burdelski M. Schaefer H. Setzer B. Walker U.A. Horvath R. Arch. Neurol. 2006; 63: 1129-1134Crossref PubMed Scopus (92) Google Scholar). The MDS may also arise from deficiencies of other enzymes involving mitochondrial nucleotide metabolism or transportation, such as the thymidine phosphorylase (TP) and the p53-controlled ribonucleotide reductase (p53R2) (27Nishino I. Spinazzola A. Hirano M. Science. 1999; 283: 689-692Crossref PubMed Scopus (748) Google Scholar, 28Bourdon A. Minai L. Serre V. Jais J.-P. Sarzi E. Aubert S. Chretien D. de Lonlay P. Paquis-Flucklinger V. Arakawa H. Nakamura Y. Munnich A. Rotig A. Nat. Genet. 2007; 39: 776Crossref PubMed Scopus (439) Google Scholar). These proteins account for just a fraction of all MDS cases and defects in other genes may also be involved in the etiology of MDS. To figure out the complete enzymatic steps of the salvage pathway for deoxyribonucleotide synthesis in mitochondria, we cloned and characterized the human homolog (hypothetical protein LOC129607, NCBI accession NP_997198) of a mouse gene, which was designated as thymidylate kinase family LPS-inducible member (TYKi) because of a putative thymidylate kinase domain (29Lee C.G. O'Brien W.E. J. Immunol. 1995; 154: 6094-6102PubMed Google Scholar). Previous studies showed that the expression of murine TYKi was induced or up-regulated by LPS and several cytokines (TNFα, IFNγ, IL-1β, IFNα) and was down-regulated by TGFβ (29Lee C.G. O'Brien W.E. J. Immunol. 1995; 154: 6094-6102PubMed Google Scholar, 30Björkbacka H. Fitzgerald K.A. Huet F. Li X. Gregory J.A. Lee M.A. Ordija C.M. Dowley N.E. Golenbock D.T. Freeman M.W. Physiol. Genomics. 2005; 19: 319-330Crossref Scopus (247) Google Scholar, 31Lund S. Christensen K.V. Hedtjarn M. Mortensen A.L. Hagberg H. Falsig J. Hasseldam H. Schrattenholz A. Porzgen P. Leist M. J. Neuroimmunol. 2006; 180: 71-87Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 32Falsig J. Porzgen P. Lund S. Schrattenholz A. Leist M. J. Neurochem. 2006; 96: 893-907Crossref PubMed Scopus (79) Google Scholar, 33Paglinawan R. Malipiero U. Schlapbach R. Frei K. Reith W. Fontana A. Glia. 2003; 44: 219-231Crossref PubMed Scopus (94) Google Scholar, 34Wang J. Campbell I.L. J. Virol. 2005; 79: 8295-8302Crossref PubMed Scopus (70) Google Scholar, 35Garvey T.L. Dyer K.D. Ellis J.A. Bonville C.A. Foster B. Prussin C. Easton A.J. Domachowske J.B. Rosenberg H.F. J. Immunol. 2005; 175: 4735-4744Crossref PubMed Scopus (51) Google Scholar), whereas there are no reports about its protein properties so far. Here, we cloned the full-length coding sequence of the human cDNA and found that the gene product was localized in the mitochondria of HeLa cells. We expressed this protein in insect cells and purified it to homogeneity. The recombinant protein phosphorylated CMP, UMP, dCMP, dUMP, and monophosphates of the pyrimidine nucleoside analogs ddC, dFdC, araC, BVDU, and FdUrd. Based on its substrate recognition and intracellular location we designated this novel enzyme as mitochondrial UMP-CMP kinase and named it UMP-CMPK2. The relative phosphorylation efficacy of the natural substrates were dUMP > dCMP > CMP > UMP, which is different from the properties of cytosolic UMP-CMPK. The characteristics and phylogenetic analysis demonstrated that this protein comes from a novel family of UMP-CMP kinases. Cloning of Human UMP-CMPK2 cDNA—For PCR amplification of UMP-CMPK2 cDNA, the ATCC image clone 3063188 (GenBank™ ID: AW408129, BC089425) (ATCC: 9556333) was used as template, the forward primer containing the encoding sequence of the first 13 amino acids of hypothetical protein LOC129607 was 5′-CAC CAT GGC CTT CGC CCG CCG GCT CCT GCG CGG GCC ACT GTC G-3′, the reverse primer was 5′-GAA GTA AAA TTA AGA TGC CTG GTC TCC AGT TTT CTG-3′. PCR was performed in an Apollo ATC201 Thermal Cycler (CLP Molecular Biology) with Platinum Pfx DNA polymerase (Invitrogen) and 2× PCRx Enhancer solution. The cycling parameters are 1× (94 °C, 2 min), 30× (94 °C, 30 s; 55 °C, 30 s; 68 °C, 2 min) and 1× (68 °C, 10 min). PCR products were cloned into pENTR™/S.D./D-TOPO entry vector (Invitrogen) with TOPO cloning method to create an entry clone pENTR-UCMPK2-E448G. The clone was verified by DNA sequencing (MWG Biotech). Phylogenetic Analysis of UMP-CMPK2—Multiple sequence alignments were accomplished with Kalign program on the EBI server and edited with the GeneDoc v2.6 program. The rooted tree was constructed with PhyML and was plotted with iTOL v1.01. Subcellular Localization of UMP-CMPK2—Two plasmids were constructed for protein expression in mammalian cells. The first plasmid was based on pEGFP-N1 vector (Clontech). Two oligos 5′-GAA TTC ATG GCC TTC GCC CGC CGG CTC CTG CGC GGG CCA CTG TCG GGG CCG CTG CTC GGG CGG CGC GGG GAT CCA-3′ and 5′-GGA TCC CCG CGC CGC CCG AGC AGC GGC CCC GAC AGT GGC CCG CGC AGG AGC CGG CGG GCG AAG GCC ATG AAT TCA-3′ were used to form a dsDNA fragment encoding the first 22 amino acids of UMP-CMPK2. This dsDNA was ligated into the EcoRI-BamHI site of pEGFP-N1 to create an expression plasmid pN22-GFP. The second plasmid was based on pcDNA-DEST47 destination vector (Invitrogen). Using pENTR-UCMPK2-E448G as template, with forward primer 5′-CAC CAT GGC CTT CGC CCG CCG GCT C-3′ and reverse primer 5′-GTG AGG ATC CGG TCC ACT AAA ACT ATT CTG GAT TAG G-3′ in which the stop codon was deleted for C-terminal fusion purpose, full-length coding region of UMP-CMPK2 cDNA was amplified by PCR under the same condition of the first PCR except the following parameters were used: 1× (94 °C, 3 min), 30× (94 °C, 15 s; 58 °C, 30 s; 68 °C, 2 min) and 1× (68 °C, 10 min). PCR products were cloned into pENTR™/S.D./d-TOPO to create an entry clone. The mammalian expression plasmid pUCMPK2-E448G-GFP was constituted by recombination of this entry clone and pcDNA-DEST47 (Invitrogen) according to the manufacturer's instructions. The HeLa cell line (American Type Culture Collection) was transfected with pN22-GFP and pUCMPK2-E448G-GFP according to the method described (19Curbo S. Amiri M. Foroogh F. Johansson M. Karlsson A. Biochem. Biophys. Res. Commun. 2003; 311: 440-445Crossref PubMed Scopus (10) Google Scholar). The cells were stained with MitoTracker Red (Molecular Probes) 48 h after transfection. Cell fluorescence was imaged with a Nikon Eclipse E600 microscope equipped with a SPORT RT digital camera. Expression and Purification of Recombinant UMP-CMPK2—The cDNA sequence without putative mitochondrial targeting signal was amplified by PCR with forward primer 5′-CAC CGG ATC CAT GGT CTG CGC TGG GGC CAT GG-3′ (with BamH I site), reverse primer 5′-GAA TTC CTA GTG ATG GTG ATG GTG ATG CGG TTC ACT AAA ACT ATT CTG-3′ (with EcoRI site and six histidine codons), Platinum Pfx DNA polymerase (Invitrogen) and 1× PCRx Enhancer solution. The cycling parameters are 1× (94 °C, 2 min), 30× (94 °C, 30 s; 58 °C, 30 s; 68 °C, 2 min) and 1× (68 °C, 10 min). PCR products were cloned into pENTR™/SD/D-TOPO to create an entry clone. The UMP-CMPK2Δ21-6His coding sequence from this entry clone was inserted into the BamHI-EcoRI site of the pBacPAK8 transfer vector (Clontech) to get a transfer construct. Recombinant virus was constructed by cotransfecting Spodoptera frugiperda (Sf9) cells with the transfer construct and BacPAK6 viral DNA according to the manufacturer's manual. Protein expression in Sf9 cells and the affinity chromatography were modified from reported protocol (36Korhonen J.A. Gaspari M. Falkenberg M. J. Biol. Chem. 2003; 278: 48627-48632Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Sf9 cells were harvested 72 h after infection with 10 p.f.u. of recombinant virus. The cells were lysed, and the extract was cleared by centrifugation at 40,000 rpm × 30 min, 4 °C, using a Beckman 45TI rotor. The recombinant proteins were purified with TALON Metal Affinity Resin (Clontech), desalted on PD-10 column (Amersham Biosciences) and followed by anion-exchange chromatography with a MonoQ HR5/5 (Amersham Biosciences) column pre-equilibrated in low salt Buffer IEX (20 mm Tris-HCl, pH 8.5, 50 mm NaCl, 10% glycerol, 1 mm DTT, and 1× protease inhibitors). After elution in a linear gradient (10 ml) of Buffer IEX (0.05–1 m NaCl), the recombinant proteins were concentrated with Centricon 10 devices (Millipore) and applied to a Superose 6 10/300 GL column (Amersham Biosciences) pre-equilibrated in Buffer GF (20 mm Tris-HCl, pH 8.0, 200 mm NaCl, 10% glycerol, and 1 mm DTT). The proteins were eluted at a flow rate of 0.30 ml/min with 1.5 bed volumes of Buffer GF. The calibration of column was carried out with Bio-Rad gel filtration standard. Isoelectric Point Determination—The pI of recombinant protein was determined on a PhastSystem with IEF PhastGel IEF 3–9 media (Amersham Biosciences) according to the manufacturer's instruction. Enzyme Assays—The nucleoside monophosphates and nucleoside analogs (Sigma) were added at a final concentration of 1 mm in a 10-μl reaction mixture containing 50 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 1 mm unlabeled ATP, 1 μCi of [γ-32P]ATP (3000 Ci/mmol) (Amersham Biosciences), and 0.1–1 μg of UMP-CMPK2Δ21-6His recombinant proteins. The reactions were carried out for 1 h (nucleoside monophosphates) or 2 h (nucleoside analogs) at 37 °C. 1 μl of reaction mixtures were spotted on poly(ethyleneimine)-cellulose F chromatography sheets (Merck Inc.), and the nucleosides were separated in 0.5 m ammonium formate, pH 3.5 (37Jean Lust M.A.S. J. Chromatogr. A. 1972; 71: 127-133Crossref Scopus (16) Google Scholar). The sheets were autoradiographed by phosphoimaging plates (BAS 1000, Fuji Photo Film). For Michaelis-Menten kinetic properties, the non-radiolabeled products were separated and quantified by reversed-phase HPLC using a Chromolith™ column (RP-18e, 100-4.6 mm) (Merck Inc.) as described before (19Curbo S. Amiri M. Foroogh F. Johansson M. Karlsson A. Biochem. Biophys. Res. Commun. 2003; 311: 440-445Crossref PubMed Scopus (10) Google Scholar). Northern Blotting—The human cancer cell line Northern blot including the mRNA from 8 different cancer cell lines was purchased from Clontech Inc. A 644-bp cDNA encoding the C-terminal domain was used as probe. Hybridization was carried out according to the manufacturer's instructions. Cloning of the Full-length cDNA and Primary Structure Analysis of the UMP-CMPK2 Protein—The human UMP-CMP kinase 2 is currently recorded as hypothetical protein LOC129607 in Entrez Gene data base of NCBI. The genomic sequence is localized at 2p25.2. It is regarded as a thymidylate kinase family LPS-inducible member because its homology with mouse TYKi protein, which was LPS-inducible in macrophages and was supposed to have thymidylate kinase activity based on sequence similarity with thymidylate kinase (29Lee C.G. O'Brien W.E. J. Immunol. 1995; 154: 6094-6102PubMed Google Scholar). Although many ESTs already have been cloned, no mRNA or cDNA that covers the full-length coding region has been characterized. To clone the full-length cDNA, we selected the ATCC cDNA clone IMAGE3063188 (NCBI accession BC089425) as our PCR template. By adding the coding sequence of 8 missing amino acids in the forward primer, we PCR amplified the full-length cDNA of UMP-CMPK2. Because this cDNA was derived from BC089425, which contains a point mutation at the codon of the 448th amino acid (GGA (codon of glycine) in BC089425 and GAA (codon of glutamic acid) in genomic DNA and all other ESTs), we designated the protein coded by this cDNA sequence as UMP-CMPK2-E448G. For protein expression, this point mutation was corrected by using the genomic sequence in reverse primers for PCR amplification. An interesting feature of the cDNA sequence is that the first half of the coding sequence has high GC content. From the translation start site, the 650-bp cDNA at the 5′-end has 76.3% GC content, whereas the 3′-end cDNA sequence only has about 52.6% GC. UMP-CMPK2 has 449 amino acids, which is longer than UMP-CMPK (228 amino acids). Primary structure analysis with Motif Scan and multiple sequence alignment with other thymidylate kinases showed that the protein sequence had a thymidylate kinase domain (Fig. 1B). The C-terminal domain contains all the consensus motifs: P-loop (ATP/GTP binding motif A), LID domain, adenine-base binding loop, catalytic site, and potential substrate binding site (Fig. 1B). Phylogenetic analysis was accomplished among selected species, which evolutionarily cover a long distance range from prokaryotes to primates (Fig. 1A). UMP-CMPK2, UMP-CMPK, thymidylate kinase (TMPK), cytidylate kinase (CMPK), and uridylate kinase (UMPK) from some prokaryotes were selected for comparison. On the phylogenetic tree, it is clear that the distance between the UMP-CMPK2 family and the TMPK family was much shorter than the distance between UMP-CMPK2 and UMP-CMPK. The predicted UMP-CMPK2 of dogs has quite low homology with other UMP-CMPK2 members (Fig. 1, A and B). Subcellular Localization of UMP-CMPK2 in HeLa Cells—Original analysis with PSORT program showed that UMP-CMPK2 had high probability of being a mitochondrial protein with a putative cleavage site of the mitochondrial targeting signal after the 20th or the 32nd amino acid. To confirm this prediction, a dsDNA fragment encoding the first 22 amino acids of UMP-CMPK2 was synthesized and cloned into the pEGFP-N1 vector to construct N22-GFP fusion gene. After transfection with the pN22-GFP plasmid, HeLa cells were cultured for ∼48 h and then were stained with MitoTracker Red. Results showed that most of the green fluorescence of GFP distributed in a typical fibrillar pattern as mitochondria usually do. Overlay of the fluorescence signal of GFP and MitoTracker Red demonstrated the fibrillar mitochondrial localization in yellow color (Fig. 2, top panel). The full-length UMP-CMPK2-E448G cDNA was fused to the GFP gene and was transiently expressed in HeLa cells as a UMP-CMPK2-E448G-GFP fusion protein. As shown in Fig. 2, bottom panel, the green signal of UMP-CMPK2-E448G-GFP was also overlaid with the red signal of MitoTracker Red, which confirmed the mitochondrial localization of UMP-CMPK2 in HeLa cells. Protein Expression and Purification of Recombinant UMP-CMPK2—At first, we thought that this protein would have TMPK activity based on BLAST results and interpretation of the mouse homolog (29Lee C.G. O'Brien W.E. J. Immunol. 1995; 154: 6094-6102PubMed Google Scholar). Initially, UMP-CMPK2 was expressed as His-tagged and GST-tagged recombinant proteins in Escherichia coli, respectively. We cleaved off the tags with protease and purified the proteins to high homogeneity (>95%). All enzymatic activity assays performed under conditions for TMPK activity only showed UMP-CMP kinase activity. No phosphorylation of dTMP was detected (data not shown). Considering a report that human TMPK activity was undetectable in transformed yeast or from proteins of several E. coli expression systems (38Huang S.-H. Tang A. Drisco B. Zhang S.-q. Seeger R. Li C. Jong A. DNA Cell Biol. 1994; 13: 461-471Crossref PubMed Scopus (49) Google Scholar), we overexpressed His-tagged recombinant UMP-CMPK2 in insect cells and obtained pure proteins for enzymatic assay. First we designed a new set of primers for PCR amplification. The forward primer started at the 22nd amino acid to remove the cleavable putative mitochondrial targeting signal. One ATG codon was added before the 22nd amino acid to start translation. The reverse primer was located around the position of stop codon, but the stop codon was replaced by a histidine codon followed by 5 more to encode the His6 tag. The E448G point mutation was also corrected to glutamic acid (Glu, E) by using the genomic sequence. The PCR products were used to construct recombinant Baculovirus carrying UMP-CMPK2Δ21-6His fusion gene. The Sf9 cells were infected with recombinant virus and harvested 3 days later. To characterize the properties of the enzyme, UMP-CMPK2Δ21-6His protein was first purified with affinity chromatography. The protein was quite pure according to the SDS-PAGE data (Fig. 3B, lane 2). To reach a higher purity we performed two more purification steps: anion-exchange and gel filtration chromatography. The final products had very high purity (>95%) as shown by SDS-PAGE (Fig. 3B) and gel filtration diagram (data not shown). The pI value of the recombinant protein was 6.05 as determined by isoelectric focusing method along with pI markers of pH 4.55–8.65 (Fig. 3A). The protein did not bind the MonoQ matrix stably at pH 8.0, especially when large amounts of proteins were loaded. Instead we used pH 8.5 for anion-exchange chromatography. Because the pH value in mitochondria is about 8.0 (39Llopis J. McCaffery J.M. Miyawaki A. Farquhar M.G. Tsien R.Y. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6803-6808Crossref PubMed Scopus (925) Google Scholar, 40Abad M.F.C. Di Benedetto G. Magalhaes P.J. Filippin L. Pozzan T. J. Biol. Chem. 2004; 279: 11521-11529Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), we kept pH 8.0 for gel filtration chromatography and the following activity assays. Gel filtration diagram showed two peaks of UMP-CMPK2. Referring to the molecular size calibration curve from gel filtration molecular size standards, the major peak was monomer, whereas the minor peak had molecular weight of ∼89 kDa indicating dimer form of the recombinant protein. Substrate Specificity of UMP-CMPK2—Substrate specificity of recombinant protein was studied with thin layer chromatography (TLC) assay by using the [γ-32P]ATP as phosphate donor. As shown in Fig. 4A, the recombinant UMP-CMPK2 could phosphorylate CMP, UMP, dCMP, and dUMP. We also investigated the phosphorylation of dCMP and dUMP analogs using a two-step enzymatic method. For dCyd analogs, the human deoxycytidine kinase (dCK) was added into each reaction to catalyze the first phosphorylation. The results showed that ddC-MP, dFdC-MP, and araC-MP can be phosphorylated by UMP-CMPK2 (Fig. 4B). For dUrd analogs, we used human TK2 for the coupled phosphorylation. BVDU-MP and FdU-MP were proved to be substrates of UMP-CMPK2 in this assay (Fig. 4C). Kinetic Properties of UMP-CMPK2—The Michaelis-Menten kinetic properties of the enzyme were determined with four natural substrates using reversed-phase HPLC (Table 1). The production of dUDP was calculated referring to the chromatography data of UDP because there was no pure dUDP standard available. UMP-CMPK2 showed preference for dUMP and dCMP compared with CMP and UMP. The dUMP was the preferred substrate and the Vmax/Km value was about 35-fold higher than that for dCMP, and about 1600-fold higher than that for the poorest substrate UMP. The differences in Vmax were about 11-fold ranging from 0.19 μmol/mg/min (UMP) to 1.77 μmol/mg/min (dCMP). The Vmax of CMP and dCMP was similar, whereas Vmax of dUMP was 2.5-fold of the maximum rate for UMP. The differences in Km were 63-fold ranging from 0.10 mm (dUMP) to 6.30 mm (UMP). There was less than 3-fold difference between the Km of CMP and dCMP (Table 1).TABLE 1Kinetic properties of recombinant human mitochondrial UMP-CMP kinase with ATP as phosphate donor All reactions were performed at 37 °C. Km values were derived from the Lineweaver-Burk plot. Vmax values were calculated using the Michaelis-Menton equation: ν = Vmax[S]/Km + [S]. Values are presented as mean ± S.D. from at least three independent experiments.SubstrateKmVmaxVmax/Kmmmμmol/mg/minCMPaThe concentration of ATP/Mg2+ was 10:10 mm3.09 ± 0.781.64 ± 0.350.53UMPbThe concentration of ATP/Mg2+ was 5:5 mm6.30 ± 1.940.19 ± 0.110.03dCMPcThe concentration of ATP/Mg2+ was 2:6 mm1.31 ± 0.651.77 ± 0.741.35dUMPbThe concentration of ATP/Mg2+ was 5:5 mm0.10 ± 0.040.48 ± 0.1248a The concentration of ATP/Mg2+ was 10:10 mmb The concentration of ATP/Mg2+ was 5:5 mmc The concentration of ATP/Mg2+ was 2:6 mm Open table in a new tab Expression of UMP-CMPK2 in Cancer Cells—A hu" @default.
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• W2014089999 title "Human UMP-CMP Kinase 2, a Novel Nucleoside Monophosphate Kinase Localized in Mitochondria" @default.
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