臺灣博碩士論文加值系統

Mst3的分子量約52kDa,是一哺乳動物細胞中屬於絲胺酸/蘇胺酸蛋白質之激酵素，相似於酵母菌中發現的激酵素Ste20，但目前對其在細胞中的生理功能尚未知。Mst3之N端具激酵素功能區域，C端為調節功能區域。初步研究中發現，在以TNF-α 或Stamospovine引起之Jarket’ cells細胞凋亡，Mst3會被切斷成40kDa（主要為激酵素功能區域）及13kDa（主要為調節功能區域）兩片段，此一被切斷之過程可被半胱胺酸水解酵素抑因子（caspase inhibitors）阻斷，而Mst3調節功能區域被切除與細胞存活有關。在試管內試驗也發現，完整的Mst3WT存在於細胞質，但是切除調節功能區的Mst3WTΔ314則主要在細胞核，而Mst3 WTΔ314的此一細胞核轉移之機制目前仍未知，但由本實驗室之研究及其他之証據顯示，Mst3在細胞淍亡中扮演著一個必要之角色。為了更進一步瞭解細胞中Mst3之生理功能及分子作用機制，一個特異性的N端Mst3抗體是極需要的。在本研究中，我們建構Mst3的表現型質體(GST- Mst3WTΔ314, GST-Mst31-85, and GST-Mst3244-313)，並表現及純化這些融和蛋白。且用GST- Mst3WTΔ314以免疫小鼠，經過融合瘤之製備及篩選後，我們獲得一個有效抗Mst3 WTΔ314蛋白質的融合瘤，稱之為2A8. 由西方轉漬之結果得知，此一融合瘤產生之抗體只特異性的辨認Mst3，對於其他與Ste20具有88％胺基酸相似之激酵素SOK1及Mst4並無陽性反應，由ELISA及西方轉漬之結果顯示此一單株抗體對於完整Mst3WT或經切割之Mst3 WTΔ314端都能專一性的辨識。目前的結果更顯示，只要1ng的GST-Mst3WTΔ314就可被單株抗體2A8偵測到，我們進一步將分別帶有Mst3WT和Mst3WTΔ314的質體轉殖到細胞中，再利用抗HA-tagged的抗體和單珠抗體2A8去偵測發現Mst3WT 和Mst3WTΔ314分別在細胞質及細胞核，我們也直接用單珠抗體2A8偵測到細胞內生性之Mst3。未來將純化並定義此一單株抗體，用來偵測組織分佈、研究內生性Mst3在細胞內所扮演的角色及在Mst3上之抗原決定位 (epitope)。

Mst3, a 52KDa protein, is one of mammalian Ste20-like serine/threonine protein kinases with unknown physiological functions. It contains a kinase domain at its N-terminus, while a regulatory domain at its C-terminus. Previous study has shown that in vitro translated Mst3 could be cleaved by cell extracts prepared from TNF-α or Staurosporine-induced apoptotic Jurkat cells. This cleavage could be specifically inhibited by caspase inhibitors. The removal of the regulatory domain of Mst3 correlates with the decreasing of cell viability. In vitro studies have also shown that full length Mst3, Mst3WT, was present predominantly in the cytoplasm, but its truncated form, Mst3Δ314 was mainly localized in the nucleus. The significance of the nuclear translocation of Mst3Δ314 is not clear so far. The results from previous studies in our laboratory and other’s strongly suggest that Mst3 may play an essential role in mediating programmed cell death. Hence, a specific antibody for the N-terminal domain of Mst3 is crucial for the study of the physiological functions and intracellular molecular mechanism of endogenous Mst3. In this project, we constructed various expression vectors for GST-Mst fusion protein, termed GST-MstWT△314, GST-Mst1-85, and GST-Mst244-313. The expression and purification of these fusion proteins were promptly carried out. The GST-MstWT△314 fusion protein was used to immunize the mice. After hybridoma preparation and screening, one effective hybridoma clone, 2A8, was obtained. The corresponding monoclonal antibody generated by this hybridoma clone was specific. It could only recognize Mst3 but not SOK1 and Mst4, two closely related Ste20-like protein kinases with 88% amino acid sequence homology, as evidenced by Western blotting. The ELISA and Western blotting also demonstrated that this monoclonal antibody can recognize both full length as well as truncated Mst3. Preliminary result showed that as low as 1 ng GST-MstWT△314 could be detected by using unpurified monoclonal antibody 2A8. Transiently expressed full length Mst3 could be detected in the cytoplasm by both HA-tagged specific antibody and monoclonal antibody 2A8, whereas, truncated Mst3, Mst3 WT△314, was located in nucleus. The similar result was also observed in HeLa cell. The capability of monoclonal antibody 2A8 in the detection of endogenous Mst3 was also studied. An endogenous Mst3 in 293T cell could be detected by the monoclonal antibody 2A8.

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Chinese abstract…………………………………………………………..I
English abstract………………………………………………………….III
Acknowledgement……………………...…………………………..………V
Content……………………………………………………………………...VI
Figures…………………………………………………………………....VII
Abbreviation…………………………………………………………...….IX
1. Introduction………………………………………………………………1
2. Materials and Methods………………………………………………….7
3. Result…………………………………………………………………….19
4. Discussion……………………………………………………………….26
References…………………………………………………………………..28
Appendix…………………………………………………………………....49

Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., Watson, J.D., Molecular Biology of the Cell, Third Edition, 1991.
Cardone, M.H., Salvesen, G.S., Widmann, C., Johnson, G, and Frisch, S.M., 〝The regulation of Anoikis: MEKK-1 activation requires cleavage by caspase〞, Cell, 90: 315-323, 1997.
Creasy, C.L., and Chernoff, J., 〝Cloning and characterization of a human protein kinase with homology to STE20〞, J. Biol. Chem, 270: 21695-21700, 1995a.
Creasy, C.L., and Chernoff, J., 〝Cloning and characterization of a member of the MST subfamily of STE20-like kinases〞, Gene, 167: 303-306, 1995b.
Cryns, V. and Yuan J., 〝Proteases to die for〞, Genes Dev, 12: 1551-1570, 1998.
Dan,I.,Watanabe,N.M.,and Kusumi, A.Trends Cell Biol., 11,220-230, 2001.
Datta, S.R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., and Greenberg, M.E., 〝Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery〞, Cell, 91: 231-241, 1997.
Ghayur, T., Hugunin, H., Talanian, R.V., Ratnofsky, S., Quinlan, C., Emoto, Y., Pandey, P., Datta, R., Huang, Y., Kharbanda, S. Allen, H., Kamen, R., Wong, W., and Kufe, D., 〝Proteolytic activation of protein kinase C δ by an ICE/CED3-like proteinase induces characteristics of apoptosis〞, J. Exp. Med, 184: 2399-2404, 1996.
Graves, J. D., Gotoh, Y., Draves, K.E., Ambeose, D., Han, D.K.M., Wright, Chernoff, J., Clark, E.A and Krebs, E.G., 〝Caspase-mediated activation and induction of apoptosis by the mammalian STE20-like kinase Mst1〞, EMBO J, 17: 2224-2234, 1998.
Harlow, E., Lane, D., Fusions. In: Harlow, E., Lane, D. (Eds.), Antibodies─A Laboratory Manual, Cold Spring Harbor Laboratory, New York, pp. 210—213, 1988a.
Harlow, E., Lane, D., Single-Cell Cloning. In: Harlow, E., Lane, D. (Eds.), Antibodies─A Laboratory, Manual Cold Spring Harbor Laboratory, New York, pp. 219—227. 1988b
Huang,C.Y.F., Wu,Y.M., Hsu,H.Y., Lee,W.S., Lai,M.D., Lu,T.J., Huang,C.L.,Leu,T.H., Shih,H.M., Fang,H.I., Robinson,D.R., Kung,H.J., and Yuan,C.J., 〝Caspase activation of mammalian sterile 20-like kinase 3 (Mst3): Nuclear translocation and induction of apoptosis〞, J. Biol. Chem, 10.1074/jbc. M202468200, July 2002.
Qian, Z., Lin, C., Espinosa, R., LeBeau, M., Rosner, M. R., 〝Cloning and Characterization of MST4, a Novel Ste20-like Kinase〞, J. Biol. Chem, 276:22439-22445, 2001.
Jacobson, M.D., Weil, M. and Raff, M.C., 〝Programmed cell death in animal development〞, Cell, 88, 347-354, 1997.
Kohler, G., Milstein, C., 〝Continuous cultures of fused cells secreting antibody of predefined specificity〞, Nature, 256, 495, 1975.
Lee, K.K., Murakawa, M., Nishida, E., Tsubuki, S., Kawashima, S., Sakamaki, K., and Yonehara, S., 〝Proteolytic activation of MST/krs, STE20-related protein kinase, by caspase during apoptosis〞, Oncogene, 16: 3029-3037, 1998.
Nicholson, D.W., Ali, A. Thornberry, N.A., Vaillancourt, J.,Ding, C.K, allant, M., Gareau, Y., Griffin, P.R., Labelle, M., Lazebnik Y.A., Munday, N.A., Raju, M., Smulson, M.E., Yamin, T.T., Yu, V.L., and Miller, D.K., 〝Identification and inhibition of the ICE/CED3 protease necessary for mammalian apoptosis〞, Nature, 376: 37-43, 1995.
Nicholson, D.W., and Thornberry, N.A., 〝Caspases: killer proteases〞, Trends Biochem. Sci, 22: 299-306, 1997.
Porter, A.G., 〝Protein translocation in apoptosis〞, Trends in Cell Biol., 9: 394-401, 1999.
Ramer, S.W., and Davis, D.W. 〝A dominant truncation allele identifies a gene, STE20, that encodes a putative protein kinases necessary for mating in Saccharomyces cerevisiae〞, Proc. Natl. Acad. Sci. USA, 90: 452-456, 1993.
Schinkmann, K. and Blenis, J.〝Cloning and characterization of a human STE20-llike protein kinase with unusual cloning requirements〞, J. Biol. Chem, 272: 28695-28703, 1997.
Tweari, M., Quan, L.T., O’Roirke, K., Desnoyers, S.,Zeng, Z., Beidler, D., and Dixit, V.M. 〝Yama/CPP32 β , a mammalian homology of CED-3, is a CrmA-inhibitable protease that cleaves the death substrate poly (ADP-ribose) polymerase〞, Cell, 81: 801-809, 1995.
Walter, B.N., Huang, Z., Jakobi, R., Tuazon, P.T., Alnemri, E.S., Litwack, G., and Traugh, J.A. 〝Cleavage and Activation of p21-activated Protein Kinase γ -PAK by CPP32 (Caspase3)〞, J. Biol. Chem., 273: 28733-28739, 1998.
Watabe, M., Kakeya, H., and Osada, H. 〝Requirement of protein kinase (krs/MST) activation for MT-21-induced apoptosis〞, Oncogene, 18: 5211-5220, 1999.
Wen, L.-P., J. Fahrni, S. Troie, J-L. Guan, K. Orth, and G. Rosen. 〝Cleavage of focal adhesion kinase by caspases during apoptosis〞, J. Biol. Chem., 272: 26056-26061, 1997.
Wolff, B., Sanglier, J.J. and Wang, Y. 〝Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA〞, Chem. Biol., 4, 139-147, 1997.
Yao,.Z., Zhou, G., Wang, X.S., Brown, A., Diener, K., Gan, H., and Tan, T-H. 〝A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway〞, J. Biol. Chem., 274: 2118-2125, 1999.