臺灣博碩士論文加值系統

在細胞中，中心體的功能相當於microtubule聚集的中心，並且調控及決定有絲分裂時microtubule的數目、極性和組成等。對於細胞有絲分裂紡錘體的形成、細胞的極性和細胞運動，中心體扮演重要的角色。在動物細胞中，中心體由一對中心粒以及周圍的雲狀物質（PCM）所構成。目前已知這些物質包括gamma-tubulin、centrin、pericentrin及ninein。
我們利用酵母菌雜交系統，以肝醣合成激酶（GSK-3β）為釣餌，成功釣出人類中心體相關蛋白hNinein。hNinein基因的序列已完成定序，其可轉譯出2096個胺基酸，分子量則為243 kDa。蛋白質結構上，有GTP binding site，且幾乎全長皆為coiled-coil domain，其中還含有四個leucine zipper domains。而人類的ninein基因與老鼠的ninein（mNinein）基因相似性達67百分比。另外我們還發現一個不同的splicing isoform稱hNinein-lm（human Ninein like mouse），其N端有EF-hand-like domain。hNinein-lm的N端有鈣離子結合區域，但是hNinein無此區域。我們發現hNinein-lm的N端可能具有transactivation region。在酵母菌雜交系統中，發現ninein C端的coiled-coil domain（CCII：1623-1936/1617-1930 a.a.）可與ninein 含一個leucine zipper 的中間coiled-coil區域（CCI：461-1193/455-1187a.a.）以及自身(CCII：1623-1936/1617-1930 a.a.)相互作用。由酵母菌免疫沉澱法實驗，得知CCI與CCII以及CCII與CCII都會互相結合。藉由酵母菌雜交系統中，蛋白質分子間與片段區域之間有相互作用，表示ninein可能自己組合作用。在gel filtration實驗中，推測在HeLa 細胞中ninein可能形成一複雜的聚合物。由以上不同的分子與區域之間的相互作用，我們可推測ninein本身可能形成多倍體的形式，且可能與中心體相關蛋白的形成與中心粒複製的調控有關。
進一步想知道ninein不同的區域片段在細胞中所在的位置，我們將ninein不同的區域片段接在GFP，在HeLa細胞中觀察其表現。我們只發現只有CCII（1623-1936/1617-1930 a.a.）可以觀察到在中心體上的表現；N端（1-478/1-472a.a.）和CCI（461-1193/455-1187a.a）則無觀察到。因為CCII domain可能含有中心體的訊號以及會與本身和CCI domain 交互作用，所以CCII domain應是一重要的domain。我們想尋找ninein 的結合蛋白來了解ninein 的功能。我們實驗室利用酵母菌雜交系統，以ninein的C 端（1186-2096/1180-2090a.a.；1623-2096/1617-2090a.a.）為釣餌，進一步去找尋與ninein結合的蛋白，包括CKIP-1，Keratin-23，Stathmin， NUDC，CGI-99等五種。這些結合蛋白都可以與ninein的coiled-coil domain (CCI、CCII) 結合。我們推測coiled-coil domain可能在蛋白之間的結合，扮演重要的角色。CKIP-1為CK-2結合蛋白，在酵母菌雜交實驗中，只會與CK-2α結合，不會與CK-2α’結合。CKIP-1為CK-2（CK-2α）的非酵素型調控者，可能可以將CK-2α帶到細胞特定的地方。Keratin-23為intermediate-filament protein的家族成員之一。其中間的區域有大區域的α-helical rod domain，與其他成員比較，此段保留性很大。Stathmin為一種調控microtubule filament系統的phosphoprotein。Stathmin是一種調控有絲分裂的microtubule-destabilizing phosphoprotein。人類NUDC（nuclear distribution C）序列也是呈現高度保留性，分別與mouse有94%相同，與rat有95%相同。可能與人類的神經傳遞系統失調有關。藉由比較蛋白質體學，CGI-99為一種新的人類基因，與線蟲在遺傳上有保留性。這五個蛋白結構中都有Coiled-coil domain 的區域。Ninein可以與這五個蛋白（CKIP-1, Keratin-23, Stathmin, NUDC, CGI-99）結合，推測其可能在細胞週期的某一時期扮演anchoring protein的角色。

The centrosome functions as the major microtubule organizing center (MTOC) of the cell and may determines the number, polarity, and organization of mitotic microtubules. It plays a key role in the formation of the mitotic spindle, cell polarity, and cell locomotion. In animal cell, the centrosome is composed of a pair of centrioles that are surrounded by a mass of amorphous pericentriolar material (PCM). The recent identification of molecular components shows that PCM may be involved in the complex of gamma-tubulin, centrin, pericentrin, ninein.
Using human glycogen synthase kinase 3β(Gsk-3β as bait in the yeast two-hybrid system, we identified a novel human centrosome associated protein, ninein (hNinein). The full length cDNA of hNinein is completely sequenced, it showed that an open reading frame encoded a 2096 amino acids protein with a predicted molecular mass of 243kDa. The features of this protein include a potential GTP binding site and a large coiled-coil domain together with four leucine zipper domains which shares 67% overall identity with the mouse Ninein (mNinein). Recently, an alternatively splicing isoform, hNinein-lm (human ninein like mouse) which contains EF-hand-like domain in its N-terminal was discovered. hNinein-lm possesses a Ca+2 binding region, whereas it isn’t found in hNinein. In yeast two-hybrid, we found N-terminal of hNinein-lm (1-50a.a.) may possess the transactivation region. Moreover, coiled-coil domain in CCII (1623-1936/1617-1930 a.a.) can interact with CCI (461-1193/455-1187 a.a.) and CCII (1623-1936/1617-1930). CCI domaininteracts with CCII domain and CCIIdomain interacts with CCII domain in yeast by immunoprecipitation. In this study, we describe different intermolecular and interdomain binding interactions that may account for the process of ninein self-assembly in yeast two-hybrid. By gel filtration assay, ninein may form a complex protein in HeLa cells.These different intermolecule and intradomain binding interactions suggest a model in which oligomers of ninein molecule may be involved in the formation of complex centrosome matrix and the regulation of a pair of centrioles replication.
To investigate the localization of three domains in cells, we expressed GFP fusion proteins containing GFPase, CCI or CCII region of human ninein in HeLa cells. We showed that only GFP-CCII domain (1623-1936/1617-1930 a.a.) can localize in the centrosome. These phenomena did not appear in GFP-N-terminal region (1-478/1-472 a.a.) and GFP-CCI (461-1193/455-1187 a.a.). Because CCII domain may possess the signal of centrosome and can interact wih CCII and CCI domains, CCII domain is an important domain of human ninein. We searched ninein interaction protein which link to the function of ninein. Our laboratory, we have used human ninein（c-terminal） as a bait in the yeast two-hybrid system, we found several ninein-interacting proteins, such as CKIP-1, Keratin-23, Stathmin, NUDC, CGI-99. These proteins can interact with CCI and CCII domain (coiled-coil domain). We suggested that coiled-coil domain play an important role in protein-protein interaction. CKIP-1 is a CK2-interacting protein, that interacts with CK2α, but not CK2α’, in the yeast two-hybrid system. CKIP-1 is a non-enzymatic regulator of one isoform of CK2 (CK2α) with a potential role in targeting CK2α to a particular cellular location. Keratin-23 is a novel member of the keratin family (intermediate-filament proteins). The central α-helical rod domain of keratin-23 is highly conserved among each group of keratins. Stathmin is a globally disordered phosphoprotein that is involved in the regulation of the microtubule (MT) filament system. Stathmin is a microtubule-destabilizing phosphoprotein that plays a critical role in the regulation of mitosis. Human NUDC (nuclear distribution C) is highly conserved and its predicted amino acid sequence shows 94% identity to mouse NudC and 95% identity to rat NudC. The role of NUDC is in human neuronal migration disorders. CGI99 is a novel human gene evolutionarily conserved in Caenorhabditis elegans by comparative proteomics. The structure of proteins possess coiled-coil domain. Ninein interacting with several proteins, such as CKIP-1, Keratin-23, Stathmin, NUDC, CGI-99 suggest ninein may play a role of anchoring protein in a stage of the cell cycle.

目錄
中文摘要----------------------------------------1
英文摘要----------------------------------------4
緒論----------------------------------------------7
材料與方法-------------------------------------15
實驗結果----------------------------------------46
結果與討論-------------------------------------54
參考文獻----------------------------------------61
圖表----------------------------------------------66

1. Andersen, S.S. (1999) Molecular characteristics of the centrosome. Int Rev Cytol,187:51-109.
2. Andersen, S.S. (2000) Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18. Trends Cell Biol, 10(7):261-267.
3.Avila, J., Ulloa, L., Gonzalez, J., Moreno, F., Diaz-Nido, J. (1994) Phosphorylation of microtubule-associated proteins by protein kinase CK2 in neuritogenesis. Cell Mol Biol Res, 40(5-6):573-579.
4. Axtell, S.M., Truong, T.M., O''Neal, K.D., Yu-Lee, L.Y. (1995) Characterization of a prolactin-inducible gene, clone 15, in T cells. Mol Endocrinol, 9(3):312-318.
5. Bailly, E., Bornens, M. (1992) Cell biology. Centrosome and cell division. Nature,355(6358):300-301
6. Balczon, R., Bao, L., and Zimmer, W.E. (1994) PCM-1, a 228-kD centrosome autoantigen with a distinct cell cycle distribution. J Cell Biol, 124(5):783-793
7. Berger, B., Wilson, D.B., Wolf, E., Tonchev, T., Milla, M., Kim, P.S. (1995) Predicting coiled coils by use of pairwise residue correlations. Proc Natl Acad Sci U S A, 92(18):8259-8263.
8. Bobinnec, Y., Khodjakov, A., Mir, L.M., Rieder, C.L., Edde, B., Bornens, M. (1998) Centriole disassembly in vivo and its effect on centrosome structure and function in vertebrate cells. J Cell Biol,143(6):1575-1589.
9. Bosc, D.G., Graham, K.C., Saulnier, R.B., Zhang, C., Prober, D., Gietz, R.D., Litchfield, D.W. (2000) Identification and characterization of CKIP-1, a novel pleckstrin homology domain-containing protein that interacts with protein kinase CK2. J Biol Chem,275(19):14295-14306.
10. Bouckson-castaing, V., Moudjou, M., Ferguson, D.J.P., Mucklow, S., Belkaid, Y., Milon, G., and Crocker, P.R. (1996) Microtubule characterization of ninein, a new coiled-coil protein of the centrosome. J Cell Sci, 109:179-190
11. Charbaut, E., Curmi, P.A., Ozon, S., Lachkar, S., Redeker, V., Sobel, A. (2001) Stathmin family proteins display specific molecular and tubulin binding properties. J Biol Chem, 276(19):16146-16154.
12. Cohen, P., Yellowlees, D., Aitken, A., Donella-Deana, A., Hemmings, B.A., Parker, P.J.(1982) Separation and characterisation of glycogen synthase kinase 3, glycogen synthase kinase 4 and glycogen synthase kinase 5 from rabbit skeletal muscle. Eur J Biochem, 124(1):21-35.
13. Crute, B.E., Van Buskirk, R.G. (1992) A casein kinase-like kinase phosphorylates beta-tubulin and may be a microtubule-associated protein. J Neurochem, 59(6):2017-2023.
14. Curmi, P.A., Andersen, S.S., Lachkar, S., Gavet, O., Karsenti, E., Knossow, M., Sobel, A. (1997) The stathmin/tubulin interaction in vitro. J Biol Chem, 272(40):25029-25036.
15. Diaz-Nido, J., Serrano, L., Mendez, E., Avila, J. (1988) A casein kinase II-related activity is involved in phosphorylation of microtubule-associated protein MAP-1B during neuroblastoma cell differentiation. J Cell Biol, 106(6):2057-2065.
16. Diviani, D., Langeberg, L.K., Doxsey, S.J., and Scott, J.D. (2000) Pericentrin anchors protein kinase A at the centrosome through a newly identified RII-binding domain. Current Biol, 10:417-420
17. Doxsey, S.J., Stein, P., Evans, L., Calarco, P.D., and Kirschner, M. (1994) Pericentrin, a highly conserved centrosome protein involved in microtubule organization. Cell, 76:639-650
18. Doxsey, S. (2001) Re-evaluating centrosome function. Nat Rev Mol Cell Biol, 2(9):688-98.
19. Dransfield, D.T., Yeh,J.L., Bradford, A.J., Goldenring, J.R. (1997) Identification and characterization of a novel A-kinase-anchoring protein (AKAP120) from rabbit gastric parietal cells. Biochem J, 322 (3):801-808.
20. Duesberg, P., Rasnick, D. (2000) Aneuploidy, the somatic mutation that makes cancer a species of its own. Cell Motil Cytoskeleton, 47(2):81-107
21. Engel, J., Kammerer, R.A. (2000) What are oligomerization domains good for? Matrix Biol, 19(4):283-288.
22. Errabolu, R., Sanders, M.A., Salisbury, J.L. (1994) Cloning of a cDNA encoding human centrin, an EF-hand protein of centrosomes and mitotic spindle poles. J Cell Sci, 107:9-16.
23. Fry, A.M., Mayor, T., Meraldi, P., Stierhof, Y.D., Tanaka, K., Nigg, E.A. (1998) C-Nap1, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycle-regulated protein kinase Nek2. J Cell Biol, 141(7):1563-1574.
24. Fuchs, E. (1996) Genetic disorders of keratins and their associated proteins. J Dermatol Sci, 13(3):181-192.
25. Fuchs, E. (1996) The cytoskeleton and disease: genetic disorders of intermediate filaments. Annu Rev Genet, 30:197-231.
26. Fukasawa, K., Choi, T., Kuriyama, R., Rulong, S., Vande Woude, G.F. (1996) Abnormal centrosome amplification in the absence of p53. Science, 271(5256):1744-1747.
27. Gavet, O., Ozon, S., Manceau, V., Lawler, S., Curmi, P., Sobel, A. (1998) The stathmin phosphoprotein family: intracellular localization and effects on the microtubule network. J Cell Sci, 111 (22):3333-3346.
28. Hatzfeld, M., Weber, K. (1990) The coiled coil of in vitro assembled keratin filaments is a heterodimer of type I and II keratins: use of site-specific mutagenesis and recombinant protein expression. J Cell Biol, 110(4):1199-1210.
29. Hesse, M., Magin, T.M., Weber, K. (2001) Genes for intermediate filament proteins and the draft sequence of the human genome: novel keratin genes and a surprisingly high number of pseudogenes related to keratin genes 8 and 18. J Cell Sci, 114(14):2569-2575.
30. Hinchcliffe, E.H., Sluder, G. (2001) Centrosome duplication: three kinases come up a winner!
Curr Biol, 11(17):698-701.
31. Hinchcliffe, E.H., Sluder, G. (2001) "It takes two to tango": understanding how centrosome duplication is regulated throughout the cell cycle. Genes, 15(10):1167-1181.
32. Hoffmann, B., Zuo, W., Liu, A., Morris, N.R. (2001) The LIS1-related protein NUDF of Aspergillus nidulans and its interaction partner NUDE bind directly to specific subunits of dynein and dynactin and to alpha- and gamma-tubulin. J Biol Chem, 276(42):38877-38884.
33. Hong, Y.R., Chen, C.H., Chang, J.H., Wang, S.K., Sy, W.D., Chou, C.K., and Howng, S.L. (2000) Biochem Biophys Acta, 1492:513-516
34. Hong, Y.R., Chen, C.H., Chuo, M.H., Liou, S.Y., and Howng, S.L. (2000) Genomic organization and molecular characterization of the human ninein gene. Biochem Biophys Res Communi, 279:989-995
35. Hunter, A.W., Wordeman, L. (2000) How motor proteins influence microtubule polymerization dynamics. J Cell Sci, 113 (24):4379-4389.
36. Karabinos, A., Schunemann, J., Parry, D.A., Weber, K. (2002) Tissue-specific co-expression and in vitro heteropolymer formation of the two small branchiostoma intermediate filament proteins A3 and B2. J Mol Biol, 316(1):127-137.
37. Karsenti, E., Vernos, I. (2001) The mitotic spindle: a self-made machine. Science, 294(5542):543-547.
38. Kellogg, D.R., Moritz, M., Alberts, B.M. (1994) The centrosome and cellular organization. Annu Rev Biochem, 63:639-674.
39. Khodjakov, A., Rieder, C.L. (2001) Centrosomes enhance the fidelity of cytokinesis in vertebrates and are required for cell cycle progression. J Cell Biol, 153(1):237-242.
40. Koch, E.A., Spitzer, R.H., Pithawalla, R.B., Castillos, F.A., Parry, D.A. (1995) Hagfish biopolymer: a type I/type II homologue of epidermal keratin intermediate filaments. Int J Biol Macromol, 17(5):283-292.
41. Kohtz, D.S., Puszkin, S. 1989 Phosphorylation of tubulin by casein kinase II regulates its binding to a neuronal protein (NP 185) associated with brain coated vesicles. J Neurochem, 52(1):285-295.
42. Krek, W., Maridor, G., Nigg, E.A. (1992) Casein kinase II is a predominantly nuclear enzyme. J Cell Biol, 116(1):43-55.
43. Kuntziger, T., Gavet, O., Manceau, V., Sobel, A., Bornens, M. (2001) Stathmin/Op18 phosphorylation is regulated by microtubule assembly. Mol Biol Cell, 12(2):437-448.
44. Lai, C.H., Chou, C.Y., Ch''ang, L.Y., Liu, C.S., Lin, W. (2000) Identification of novel human genes evolutionarily conserved in Caenorhabditis elegans by comparative proteomics. Genome Res, 10(5):703-713.
45. Litchfield, D.W., Bosc, D.G., Canton, D.A., Saulnier, R.B., Vilk, G., Zhang, C. (2001) Functional specialization of CK2 isoforms and characterization of isoform-specific binding partners. Mol Cell Biochem, 227:21-29.
46. Lupas, A., Van Dyke, M., Stock, J.( 1991) Predicting coiled coils from protein sequences. Science, 252(5010):1162-1164.
47. Marx, J. (2001) Cell biology. Do centrosome abnormalities lead to cancer? Science, 292(5516):426-429.
48. Matsumoto, N., Ledbetter, D.H. (1999) Molecular cloning and characterization of the human NUDC gene. Hum Genet, 104(6):498-504.
49. Mayor, T., Meraldi, P., Stierhof, Y.D., Nigg, E.A., Fry, A.M. (1999) Protein kinases in control of the centrosome cycle. FEBS Lett., 452(1-2):92-95.
50. Mogensen, M.M. (1999) Microtubule release and capture in epithelial cells. Biol Cell, 91:331-341
51. Mogensen, M.M., Malik, A., Piel, M., Bouckson-castaing, V., and Bornens, M. (2000) Microtubule minus-end anchorage at centrosomal and non-centrosomal sites: the role of ninein. J Cell Sci, 113:3013-3023
52. Mogensen, M.M.(1999) Microtubule release and capture in epithelial cells. Biol Cell, 91(4-5):331-341.
53. Morris, S.M., Yu-Lee, L.Y. (1998) Expression of RNUDC, a potential nuclear movement protein, in mammalian cells: localization to the Golgi apparatus. Exp Cell Res, 238(1):23-32.
54. Oakley, C.E., Oakley, B.R. (1989) Identification of gamma-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans. Nature, 338(6217):662-664.
55. Ou, Y., and Rattner, J.B. (2000) A subset of centrosomal proteins are arranged in a tubular conformation that is reproduced during centrosome duplication. Cell Motil Cytoskeleton, 47:13-24
56. Paintrand, M., Moudjou, M., Delacroix, H., and Bornens, M. (1992) Centrosome organization and centriole architecture: their sensitivity to divalent cations. J Struc Biol, 108:107-128
57. Pang, Y.Y., Schermer, A., Yu, J., Sun, T.T. (1993) Suprabasal change and subsequent formation of disulfide-stabilized homo- and hetero-dimers of keratins during esophageal epithelial differentiation. J Cell Sci, 104 ( 3):727-740.
58. Paoletti, A., Bornens, M. (1997) Organisation and functional regulation of the centrosome in animal cells. Prog Cell Cycle Res, 3:285-299.
59. Picton, C., Woodgett, J., Hemmings, B., Cohen, P. (1982) Multisite phosphorylation of glycogen synthase from rabbit skeletal muscle. Phosphorylation of site 5 by glycogen synthase kinase-5 (casein kinase-II) is a prerequisite for phosphorylation of sites 3 by glycogen synthase kinase-3. FEBS Lett, 150(1):191-196.
60. Piel, M., Meyer, P., Khodjakov, A., Rieder, C.L., and Bornens, M. (2000) The respective contributions of the mother and daughter centrioles to centrosome activity and behavior in vertebrat cells. J Cell Biol, 149(2):317-329
61. Piel, M., Nordberg, J., Euteneuer, U., Bornens, M. (2001) Centrosome-dependent exit of cytokinesis in animal cells. Science, 291(5508):1550-1553.
62. Pihan, G.A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L., Doxsey, S.J. (2001) Centrosome defects can account for cellular and genetic changes that characterize prostate cancer progression. Cancer Res, 61(5):2212-2219.
63. Salas, P.J. (1999) Insoluble gamma-tubulin-containing structures are anchored to the apical network of intermediate filaments in polarized CACO-2 epithelial cells. J Cell Biol, 146(3):645-658.
64. Srrano, L., Diaz-Nido, J., Wandosell, F., Avila, J. (1987) Tubulin phosphorylation by casein kinase II is similar to that found in vivo. J Cell Biol, 105(4):1731-1739.
65. Serrano, L., Hernandez, M.A., Diaz-Nido, J., Avila, J. (1989) Association of casein kinase II with microtubules. Exp Cell Res, 181(1):263-272.
66. Steinert, P.M., North, A.C., Parry, D.A. (1994) Structural features of keratin intermediate filaments. J Invest Dermatol, 103:19-24.
67. Takahashi, M., Mukai, H., Oishi, K., Isagawa, T., Ono, Y. (2000) Association of immature hypophosphorylated protein kinase cepsilon with an anchoring protein CG-NAP.J Biol Chem, 275(44):34592-34596.
68. Takahashi, M., Shibata, H., Shimakawa, M., Miyamoto, M., Mukai, H., Ono, Y. (1999) Characterization of a novel giant scaffolding protein, CG-NAP, that anchors multiple signaling enzymes to centrosome and the golgi apparatus. J Biol Chem, 274(24):17267-17274.
69. Wang, J., Dreyfuss, G. ( 2001) Characterization of functional domains of the SMN protein in vivo. J Biol Chem, 276(48):45387-45393.
70. Whitehead, C.M., Rattner, J.B. (1998) Expanding the role of HsEg5 within the mitotic and post-mitotic phases of the cell cycle. J Cell Sci, 111 (17):2551-2561.
71. Woolfson, D.N., Alber, T. (1995) Predicting oligomerization states of coiled coils. Protein Sci, 4(8):1596-1607.
72. Woodgett, J.R., Cohen, P. (1984) Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase-5). Biochim Biophys Acta, 788(3):339-347.
73. Wu, K.C., Bryan, J.T., Morasso, M.I., Jang, S.I., Lee, J.H., Yang, J.M., Marekov, L.N., Parry, D.A., Steinert, P.M. (2000) Coiled-coil trigger motifs in the 1B and 2B rod domain segments are required for the stability of keratin intermediate filaments. Mol Biol Cell, 11(10):3539-3558.
74. Zhang, J.S., Wang, L., Huang, H., Nelson, M., Smith, D.I. (2001) Keratin 23 (K23), a novel acidic keratin, is highly induced by histone deacetylase inhibitors during differentiation of pancreatic cancer cells. Genes Chromosomes Cancer, 30(2):123-135.