臺灣博碩士論文加值系統

人類胸腺嘧啶激?（human thymidine kinase, hTK）負責dTTP合成，是回收路徑中重要的酵素。先前的研究已知，在G1時期hTK量相當低而在S時期其量會升高。當正常細胞處於G2/M時期，hTK會大量且快速減少，但是對於TK的降解機制，目前所知仍有限，本篇論文的主要目的在於尋找何機制促成TK的降解，並評估TK降解所須之序列訊息。
本實驗室早期研究中顯示，正常人類纖維母細胞（IMR-90）於血清刺激24小時至36小時之間，TK有降解作用發生；同樣的，在停滯於G2/M時期的細胞進入下一個G1時期的過程中，TK亦有降解作用發生。而加入proteasome 專一性抑制劑（LLnL）可減緩該降解作用，表示TK的降解作用很可能是透過Proteasome-dependent proteolysis而進行。
由於酵母菌細胞擁有多項優點，我們以裂殖酵母菌作為研究hTK的模型系統。利用酵母菌可調性之啟動子（nmt1），在加入 thiamine後可阻斷該啟動子之活化，以控制hTK在酵母菌之蛋白表現而能研究其降解機制。發現當裂殖酵母細胞由G2/M時期過渡至G1時期時，hTK發生急遽之降解作用，且較高溫之培養條件可促進hTK的降解速率。除此之外，當變異型hTK(Ser13aAla)被表現於此裂殖酵母細胞中，其穩定度相當高並累積於細胞中；相對的，另一變異型hTK(Ser13aAsp)則較野生型TK不穩定。這些結果顯示，hTK之Ser13可能參與其降解機制。最後我們將hTK表現於具溫度敏感性而有proteasome、SCF-mediated proteolysis或APC-mediated proteolysis缺陷的出芽酵母細胞中，在proteasome-defective出芽酵母細胞中，hTK的降解速率減緩。綜合實驗結果，我們可知，hTK表現於酵母菌之降解現象亦具其細胞週期性調節機制。

Human thymidine kinase ( hTK ), is a salvage enzyme for dTTP synthesis. It has been found that the level of hTK is high in the S phase but low in the G1 phase. The expression of hTK protein decreases dramatically during G2/M phase in normal cells, but the mechanism involved in its degradation is still unknown. The purpose of this study is to investigate the mechanism responsible for the degradation of hTK in the cell cycle, and to define the sequence signal involved in the degradation of hTK.
In our early studies, we found that the level of hTK in normal human fibroblast cell increases in response to serum stimulation and declines between the 24th to 36th hours after stimulation. Here, we showed that the decrement of hTK polypeptide could be inhibited with the treatment of proteasome inhibitor (LLnL), suggesting that the degradation of hTK is through a proteasome-dependent pathway.
Because yeast cells manifests an excellent model system for degradation study, we then utilized the Schizosaccharomyces pombe as a system for investigating the degradation of hTK. The expression of hTK in S. pombe was under the control of the nmt1 promoter, which can be shut off by thiamine. We found that hTK degraded efficiently during the G2/M transition in fission yeast. In addition, high temperature promoted the degradation rate of hTK. Furthermore, when a mutant type of TK carrying a mutation in Ser13aAla was expressed in this strain, it appeared to be very stable and accumulated in the yeast cells. In contrast, the expression of hTK mutant（Ser13aAsp）was barely detectable. These results indicated that Ser13 might be involved in the mechanism regulating hTK degradation. Finally, we expressed hTK in the temperature sensitive mutants of Saccharomyces cerevisiae that were defective in proteasome-dependet, SCF-mediated or APC-mediated proteolysis. We found that the degradation rate of hTK becomes slower in proteasome-defective cells. Overall, here we established that hTK undergoes a cell-cycle dependent degradation when expresses in the yeast system.

中文摘要 I
英文摘要 III
縮寫表 V
緒論 1
材料與方法 11
實驗結果 27
討論 36
圖表 41
參考文獻 60

1. Alfa, C., Fantes, P., Hyams, J., McLeod, M., Warbrick, E. ( 1993 ) Experiments with fission yeast. P. 95-98.（Cold Spring Habor Labotary）.
2. Bello, L. J. ( 1974 ) Regulation of thymidine kinase synthesis in human cells. Exp. Cell Res. 89：263-274.
3. Bradshaw, H. D., Jr. (1983) Molecular cloning and cell cycle regulation of a functional human tymidine kinase. Proc. Natl. Acad. Sci. USA 80：5588-5591.
4. Bradshaw, H. D., Jr and Deininger, P. I. ( 1984 ) Human thymidine kinase gene：molecular cloning and nucleotide sequence of a cDNA expressible in mammalin cells. Mol. Cell. Biol. 4：2316-2320.
5. Brent, T. P. ( 1971 ) Periodicity of DNA synthetic enzymes during the HeLa cell cycle. Cell Tiss. Kinet. 4：297-305.
6. Chang Z. F., and Huang, D. Y. ( 1993 ) The regulation of thymidine kinase in HL-60 human promyeloleukemia cells. J. Biol. Chem. 268：1266-1271.
7. Chang, B., Sen, P., Hofmann, K., Ma, L., Goebl, M., Hrper, J.W., and Elledge, S. J. ( 1996 ) SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86：263-274.
8. Chang, Z. F., Huang, D. Y., and Chi, L. M. ( 1998 ) Serine 13 is the site of mitotic phosphorylation of human thymidnine kinase. J. Biol. Chem. 273：12095-12100.
9. Chang, Z. F., Huang, D. Y., and Hsue, N. C. ( 1994 ) Differential phosphrylation of human thymidine kinase in proliferating and M phase-arrested human cells. J. Biol. Chem. 269：21249-21254.
10. Ciechanover A. ( 1998 ) The ubiquitin-proteasome pathway: on protein death and cell life. EMBO J. 17：7151-7160.
11. Ciosk, R., Zachariae, W., Michaelis, C., Shevchenko, A., Mann, M., and Nasmyth, K. ( 1998 ) An ESP1/PDS1 complex regulates loss of sister chromatid cohesion at the metaphase to anaphase transition in yeast. Cell 93：1067-1076.
12. Coppock, D., and Pardee, A. B. ( 1987 ) Control of thymidine kinase mRNA during the cell cycle. Mol. Cell. Biol. 7：2925-2932.
13. Dou, Q-P., Markell, P. J., and Pardee, A. B. ( 1992 ) Thymidine kinase transcription is regulated at G1/S by a complex that contains retinoblastoma-like protein and a cdc2 kinase. Proc. Natl. Acad. Sci. USA 89：3256-3260.
14. Elesvir, S. M., Kucherlappati, R. S., Nichols, E. A., Creagag, R. E., Ruddle, F. H., Willecke, K. and McDougall, J. K. ( 1974 ) Assinment of the gene for galactokinase to human chromosome 17 and its regional locolization to band g21-22. Nature 251：633-635.
15. Furnari, B., Blasina, A., Boddy, M. N., McGowan, C. H., and Russell, P. ( 1999 ) Cdc25 inhibited in vivo and in vitro by checkpoint kinase Cds1 and Chk1. Mol. Biol. Cell 10：833-845.
16. Groll, M., Ditzel, L., Lowe, J., Stock, D., Bochtler, M., Bartunik, H. D., and Huber, R. ( 1997 ) Structure of the 20S proteasome from yeast at 2.4 A resolution. Nature 386：463-471.
17. Gross, M.K., and Merrill, G. F. ( 1989 ) Thymidine kinase synthesis is repressed in nonreplicating muscle cells by a translational mechanism that does not affect the polysomal distribution of thymidine kinase mRNA. Proc. Natl. Acad. Sci. USA 86：4987-4991.
18. Hershko, A., and Ciechanover, A. ( 1998 ) The ubiquitin system. Annu. Rev. Biochem. 67：425-479.
19. Hershko, A., Heller, H., Elias, S., and Ciechanover, A. ( 1983 ) Components of ubiquitin-protein ligase system. Regulation, affinity purification, and role in protein breakdown. J. Biol. Chem. 258：8206-8214.
20. Ito, M., and Conrad, S. E. ( 1990 ) Independent regulation of thymidine kinase mRNA and enzyme levels in serum stimulated cells. J. Biol. Chem. 265：6954-6960.
21. Johansson, M., Karlsson, A., ( 1997 ) Cloning of the cDNA and chromosome locolization of the gene for human thymidine kinase 2. J. Biol. Chem. 272：8454-8458.
22. Kauffman, M. G., and Kelly, T. J. ( 1991 ) Cell cycle regulation of thymidine kinase： residues near the carboxyl terminus are essential for the specific degradation of the enzyme at mitosis. Mol. Cell. Biol. 11：2538-2546.
23. Kim, Y. K., and Lee, A. S. ( 1992 ) Identification of a protein-binding site in the promoter of the human thymidine kinase gene required for the G1-S regulated transcription. J. Biol. Chem. 267：2723-2727.
24. Kotani, S., Tugendreich, S., Fujii, M., and Jorgensen, PM. ( 1998 ) PKA and MPF-activted polo-like kinase regulate anaphase- promoting complex activity and mitosis progression. Mol. Cell 1：371-380.
25. Laskey, R. A., Fairman, M. P., and Blow, J. J. ( 1989 ) S phase of the cell cycle. Science 246：609-613.
26. Lau, Y.-F., and Kan, Y.-W. (1984 ) Direct isolation of the functional human thymidine kinase gene with a cosmid shuttle vector. Proc. Acad. Natl. Sci. USA 81：414-418.
27. Li, L-J., Naeve, G. S., and Lee, A. S. ( 1993 ) Temporal regulation of cyclin A p107 and p33cdk2 complex binding to a human thymidine kinase promoter element important for G1-S phase transcriptional regulation. Proc. Natl. Acad. Sci. USA 90；3554-3558.
28. Lieberman, H. B., Lin, P. F., Yeh, D. B., and Ruddle, F. H. ( 1988 ) Transcriptional and post-transcriptional mechanisms regulate murine tyhmidine kinase gene expression in serum stimulated cells. Mol. Cell. Biol. 8：5280-5291.
29. Lin, P.-F., Zhao, S.-Y. and Ruddle, F. H. ( 1983 ) Genomic cloning and preliminary characterization of the human thymidine kinase gene. Proc. Natl. Acad. Sci. USA 80：6528-6532.
30. McDugall, J. K., Kucherlapati, R. and Ruddle, F. H. (1973 ) Locolization and induction of human thymidine kinase gene by adenovirus 12. Nature New Biol. 245：172-175.
31. Motagnoli, A., Fiore, F., Evtan, E., Carrano, AC., Draetta, GF., Hershko, A., and Pangano, M. ( 1999 ) Ubiquitination of p27 is regulated by Cdk-dependent phosphorylation and trimeric complx formation. Genes Dev. 13：1181-1189.
32. Muller, R., Mumberg, D., and Lucibello, F. C. ( 1993 ) Signals and genes in control of cell-cycle progression. Biochem. Biophys. Acta. 1155：151-179.
33. Murray, A. W., and Kirschner, M. W. ( 1989 ) Dominoes and clock：the union of two views of the cell cycle. Science 246：614-621.
34. Nigg, EA. (1998 ) Polo-like kinase : positive regulators of cell division from start to finish. Curr. Opinin Cell Biol. 10：776-783.
35. Okazaki, K., Okazaki, N., Kume, K., Jinno, S., Tanaka, K., and Okayama, H. ( 1990 ) High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans- complementation of Schizosacchromyces pombe. Nucleic Acids Res. 18：6485-6489.
36. Pardee, A. B. ( 1989 ) G1 events and regulation of cell proliferation. Science 246：603-608.
37. Patton EE., Willems AR., Sa, D., Kuras, L., Thomas, D., Craig, KL., and Tyer, M. ( 1998 ) Cdc53 is a scaffold protein for multiple Cdc34/Skp1/F-box protein complex that regulate cell division and methionine biosynthesis in yeast. Genes Dev. 12：692-705.
38. Patton, E. E., Willems, A. R., and Tyer, M. ( 1998 ) Combinatorial control in ubiquitin-dependent proteolysis：don''t Skp the F-box hypothesis. Trends Genet. 4：236-243.
39. Pegoraro, L. and Bernengo, M. G. ( 1971 ) Thymidine kinase deoxycytidine kinase and deoxycytidylate deaminase activities in phytohaemagglutinin stimulated human lymphocyte. Exp. Cell Res. 68：283-290.
40. Rusell, A., Thompson, M. A., Hendley, J., Trute, L., Armes, J., and Germain, D. ( 1999 ) Cyclin D1 and D3 associate with the SCF complex and coordinately elevated in breast cancer. Oncogene 18：1983-1991.
41. Scheffner, M., Nuber, U., and Huibregtse, J.M. ( 1995 ) Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 373：81-83
42. Schneider, Yang, Q.-H., and Futcher, A. B. ( 1996 ) Linkage of replication to start by the Cdk inhibitor Sic1. Science. 272：560-562.
43. Schwob, E., Bobm, T., Mendenhall, M. D., and Nasmyth, K. ( 1994 ) The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae. Cell 79：233-244.
44. Sherley, J. L., and Kelly, T. ( 1988 ) Regulation of human thymidine kinase during the cell cycle. J. Biol. Chem. 263：8350-8358.
45. Shteinberg, M., Protopopov, Y., Listovsky, T., Brandesis, M., and Hershko, A. ( 1999 ) Phosphorylation of the cyclosome is required for its stimulation by Fizzy/cdc20. Biochem. Biophys. Res. Commun. 260：1293-198.
46. Stewart, C. J., Ito, M., and Conard, S. E. ( 1987 ) Evidence for transcriptional and post-transcriptional control of the cellular thymidine kinase gene. Mol. Cell. Biol. 7：1156-1163.
47. Stuart, P., Ito, M., Stewart, C. J.,and Conrad, S. E. ( 1985 ) Induction of cellular thymidine kinase occurs at mRNA level. Mol. Cell. Biol. 5：1490-1497.
48. Sutterluety, H., Bart, S., Karlseder, J., Wintersberger, E., and Seiser, C. ( 1996 ) Carboxy-terminal residues of mouse thymidine kinase are essential for rapid degradation in quiescent cells. J. Mol. Boil. 259：383-392.
49. Sutterluty, H., Chatelain, E., Marti, A., Wribelauer, C., Senften, M., Muller, U., and Krek, W. ( 1999 ) p45Skp2 promotes p27Kip1 degradation and induces S phase in quiesencent cells. Nat. Cell Biol. 1：207-214.
50. Taylor, A. T., Stafford, M. A. and Jones, O. W. ( 1972 ) Properties of thymidine kinase partially purified from human fetal and adult tissues. J. Biol. Chem. 247：1930-1935.
51. Travali, S., Lipson, K., Jaskulski, D., Laurent, E., and Baserga, R. ( 1988 ) Role of the promoter in the regulation of the thymidine kinase gene. Mol. Cell. Biol. 8：1551- 1557.
52. Tyer, M. ( 1996 ) The cyclin-dependent kinase inhibitor p40SIC1 imposes the requirment for Cln G1 cyclin function at Start. Proc. Nat. Acad. Sci. USA 93：7772-7776.
53. Tyers, M., and Jorgensen, P. ( 2000 ) Proteolysis and the cell cycle : with this RING I do thee destroy. Curr. Opin. Gene. & Dev. 10：54-64.
54. Tyers, M., and Willems, A.R. ( 1999 ) One ring to rule a superfamily of E3 ubiquitin ligases. Science 284：601-604.
55. Zachariae W., and Nasmyth, K. ( 1999 ) Whose end is destruction : cell division and the anaphase-promoting complex. Genes Dev. 13：2039-2058.
56. Zachariae, W., Schwab, M., Nasmyth, K., and Seufert, W. ( 1998 ) Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex. Science 282：1721-1724.