Abstract
The level of human thymidine kinase (TK) polypeptide is subject to cell cycle regulation. The enzyme is barely detectable in G1 phase but increases 10- to 20-fold by M phase. The low level of human TK in G1 phase is due primarily to the specific degradation of the protein during cell division. Substitution of heterologous promoters, removal of the introns, and deletion of all of the 3' untranslated region from the human TK gene do not affect cell cycle regulation of the enzyme. However, deletion of the carboxyl-terminal 40 amino acids or fusion of beta-galactosidase to the carboxyl terminus of human TK completely abolishes cell cycle regulation and stabilizes the protein throughout the cell cycle. These alterations do not significantly alter the specific enzymatic activity of TK. Changing the carboxyl terminus or deletion of the last 10 amino acids does not alter cell cycle regulation. These data demonstrate that residues near the carboxyl terminus of TK are essential for the cell cycle phase-specific degradation of the enzyme.
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- Bello L. J. Regulation of thymidine kinase synthesis in human cells. Exp Cell Res. 1974 Dec;89(2):263–274. doi: 10.1016/0014-4827(74)90790-3. [DOI] [PubMed] [Google Scholar]
- Booher R. N., Alfa C. E., Hyams J. S., Beach D. H. The fission yeast cdc2/cdc13/suc1 protein kinase: regulation of catalytic activity and nuclear localization. Cell. 1989 Aug 11;58(3):485–497. doi: 10.1016/0092-8674(89)90429-7. [DOI] [PubMed] [Google Scholar]
- Bradshaw H. D., Jr, Deininger P. L. Human thymidine kinase gene: molecular cloning and nucleotide sequence of a cDNA expressible in mammalian cells. Mol Cell Biol. 1984 Nov;4(11):2316–2320. doi: 10.1128/mcb.4.11.2316. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradshaw H. D., Jr Molecular cloning and cell cycle-specific regulation of a functional human thymidine kinase gene. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5588–5591. doi: 10.1073/pnas.80.18.5588. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Brent T. P. Periodicity of DNA synthetic enzymes during the HeLa cell cycle. Cell Tissue Kinet. 1971 Jul;4(4):297–305. doi: 10.1111/j.1365-2184.1971.tb01541.x. [DOI] [PubMed] [Google Scholar]
- Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chiang H. L., Dice J. F. Peptide sequences that target proteins for enhanced degradation during serum withdrawal. J Biol Chem. 1988 May 15;263(14):6797–6805. [PubMed] [Google Scholar]
- Dice J. F. Molecular determinants of protein half-lives in eukaryotic cells. FASEB J. 1987 Nov;1(5):349–357. doi: 10.1096/fasebj.1.5.2824267. [DOI] [PubMed] [Google Scholar]
- Draetta G., Beach D. Activation of cdc2 protein kinase during mitosis in human cells: cell cycle-dependent phosphorylation and subunit rearrangement. Cell. 1988 Jul 1;54(1):17–26. doi: 10.1016/0092-8674(88)90175-4. [DOI] [PubMed] [Google Scholar]
- Evans T., Rosenthal E. T., Youngblom J., Distel D., Hunt T. Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell. 1983 Jun;33(2):389–396. doi: 10.1016/0092-8674(83)90420-8. [DOI] [PubMed] [Google Scholar]
- Flemington E., Bradshaw H. D., Jr, Traina-Dorge V., Slagel V., Deininger P. L. Sequence, structure and promoter characterization of the human thymidine kinase gene. Gene. 1987;52(2-3):267–277. doi: 10.1016/0378-1119(87)90053-9. [DOI] [PubMed] [Google Scholar]
- Gross M. K., Kainz M. S., Merrill G. F. Introns are inconsequential to efficient formation of cellular thymidine kinase mRNA in mouse L cells. Mol Cell Biol. 1987 Dec;7(12):4576–4581. doi: 10.1128/mcb.7.12.4576. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Heck M. M., Hittelman W. N., Earnshaw W. C. Differential expression of DNA topoisomerases I and II during the eukaryotic cell cycle. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1086–1090. doi: 10.1073/pnas.85.4.1086. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ito M., Conrad S. E. Independent regulation of thymidine kinase mRNA and enzyme levels in serum-stimulated cells. J Biol Chem. 1990 Apr 25;265(12):6954–6960. [PubMed] [Google Scholar]
- Kauffman M. G., Noga S. J., Kelly T. J., Donnenberg A. D. Isolation of cell cycle fractions by counterflow centrifugal elutriation. Anal Biochem. 1990 Nov 15;191(1):41–46. doi: 10.1016/0003-2697(90)90384-l. [DOI] [PubMed] [Google Scholar]
- Kit S., Kit M., Qavi H., Trkula D., Otsuka H. Nucleotide sequence of the herpes simplex virus type 2 (HSV-2) thymidine kinase gene and predicted amino acid sequence of thymidine kinase polypeptide and its comparison with the HSV-1 thymidine kinase gene. Biochim Biophys Acta. 1983 Nov 17;741(2):158–170. doi: 10.1016/0167-4781(83)90056-8. [DOI] [PubMed] [Google Scholar]
- Kreidberg J. A., Kelly T. J. Genetic analysis of the human thymidine kinase gene promoter. Mol Cell Biol. 1986 Aug;6(8):2903–2909. doi: 10.1128/mcb.6.8.2903. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kwoh T. J., Engler J. A. The nucleotide sequence of the chicken thymidine kinase gene and the relationship of its predicted polypeptide to that of the vaccinia virus thymidine kinase. Nucleic Acids Res. 1984 May 11;12(9):3959–3971. doi: 10.1093/nar/12.9.3959. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewis J. A., Matkovich D. A. Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences. Mol Cell Biol. 1986 Jun;6(6):2262–2266. doi: 10.1128/mcb.6.6.2262. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lewis J. A. Structure and expression of the Chinese hamster thymidine kinase gene. Mol Cell Biol. 1986 Jun;6(6):1998–2010. doi: 10.1128/mcb.6.6.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lieberman H. B., Lin P. F., Yeh D. B., Ruddle F. H. Transcriptional and posttranscriptional mechanisms regulate murine thymidine kinase gene expression in serum-stimulated cells. Mol Cell Biol. 1988 Dec;8(12):5280–5291. doi: 10.1128/mcb.8.12.5280. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lin P. F., Lieberman H. B., Yeh D. B., Xu T., Zhao S. Y., Ruddle F. H. Molecular cloning and structural analysis of murine thymidine kinase genomic and cDNA sequences. Mol Cell Biol. 1985 Nov;5(11):3149–3156. doi: 10.1128/mcb.5.11.3149. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Littlefield J. W. The periodic synthesis of thymidine kinase in mouse fibroblasts. Biochim Biophys Acta. 1966 Feb 21;114(2):398–403. doi: 10.1016/0005-2787(66)90319-4. [DOI] [PubMed] [Google Scholar]
- Merrill G. F., Harland R. M., Groudine M., McKnight S. L. Genetic and physical analysis of the chicken tk gene. Mol Cell Biol. 1984 Sep;4(9):1769–1776. doi: 10.1128/mcb.4.9.1769. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Murray A. W., Solomon M. J., Kirschner M. W. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature. 1989 May 25;339(6222):280–286. doi: 10.1038/339280a0. [DOI] [PubMed] [Google Scholar]
- Parsell D. A., Silber K. R., Sauer R. T. Carboxy-terminal determinants of intracellular protein degradation. Genes Dev. 1990 Feb;4(2):277–286. doi: 10.1101/gad.4.2.277. [DOI] [PubMed] [Google Scholar]
- Peden K. W., Charles C., Sanders L., Tennekoon G. I. Isolation of rat Schwann cell lines: use of SV40 T antigen gene regulated by synthetic metallothionein promoters. Exp Cell Res. 1989 Nov;185(1):60–72. doi: 10.1016/0014-4827(89)90037-2. [DOI] [PubMed] [Google Scholar]
- Pines J., Hunter T. Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2. Cell. 1989 Sep 8;58(5):833–846. doi: 10.1016/0092-8674(89)90936-7. [DOI] [PubMed] [Google Scholar]
- Piper A. A., Tattersall M. H., Fox R. M. The activities of thymidine metabolising enzymes during the cell cycle of a human lymphocyte cell line LAZ-007 synchronised by centrifugal elutriation. Biochim Biophys Acta. 1980 Dec 15;633(3):400–409. doi: 10.1016/0304-4165(80)90198-1. [DOI] [PubMed] [Google Scholar]
- Riabowol K., Draetta G., Brizuela L., Vandre D., Beach D. The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells. Cell. 1989 May 5;57(3):393–401. doi: 10.1016/0092-8674(89)90914-8. [DOI] [PubMed] [Google Scholar]
- Sherley J. L., Kelly T. J. Human cytosolic thymidine kinase. Purification and physical characterization of the enzyme from HeLa cells. J Biol Chem. 1988 Jan 5;263(1):375–382. [PubMed] [Google Scholar]
- Sherley J. L., Kelly T. J. Regulation of human thymidine kinase during the cell cycle. J Biol Chem. 1988 Jun 15;263(17):8350–8358. [PubMed] [Google Scholar]
- Stewart C. J., Ito M., Conrad S. E. Evidence for transcriptional and post-transcriptional control of the cellular thymidine kinase gene. Mol Cell Biol. 1987 Mar;7(3):1156–1163. doi: 10.1128/mcb.7.3.1156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stubblefield E., Mueller G. C. Thymidine kinase activity in synchronized HeLa cell cultures. Biochem Biophys Res Commun. 1965 Aug 16;20(4):535–538. doi: 10.1016/0006-291x(65)90613-3. [DOI] [PubMed] [Google Scholar]
- Travali S., Lipson K. E., Jaskulski D., Lauret E., Baserga R. Role of the promoter in the regulation of the thymidine kinase gene. Mol Cell Biol. 1988 Apr;8(4):1551–1557. doi: 10.1128/mcb.8.4.1551. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Whitfield W. G., Gonzalez C., Maldonado-Codina G., Glover D. M. The A- and B-type cyclins of Drosophila are accumulated and destroyed in temporally distinct events that define separable phases of the G2-M transition. EMBO J. 1990 Aug;9(8):2563–2572. doi: 10.1002/j.1460-2075.1990.tb07437.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wold M. S., Kelly T. Purification and characterization of replication protein A, a cellular protein required for in vitro replication of simian virus 40 DNA. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2523–2527. doi: 10.1073/pnas.85.8.2523. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wold M. S., Weinberg D. H., Virshup D. M., Li J. J., Kelly T. J. Identification of cellular proteins required for simian virus 40 DNA replication. J Biol Chem. 1989 Feb 15;264(5):2801–2809. [PubMed] [Google Scholar]