Tau為一種微管結合蛋白（microtubule-associated protein，MAP），主要表現在神經細胞中，其功能已被證明與微管動力學（microtubule dynamic）有關。結構分析結果顯示，Tau蛋白主要由N端的projection domain及C端的microtubule-binding domain組成。功能上，projection domain已被證明與Tau的調控有關；另一方面，microtubule-binding domain則被認為和促進微管形成有關。Tau 的功能可受磷酸化和去磷酸化調控。而在Alzheimer’s disease （AD）病人腦中，由於不正常地過渡活化蛋白質激酶（kinase），造成Tau蛋白高度磷酸化，及Tau和微管崩解，並聚集形成paired helical filaments（PHFs）。在已知會磷酸化Tau的蛋白質激酶中，cdc2與GSK3β的磷酸化Tau之氨基酸位點（phosphorylation site）均被證明與PHF之磷酸化位置相同，由於cdc2與GSK3β在AD 病人腦中亦被指出有被活化的現象，因此合理推測cdc2與GSK3β為可能造成Tau蛋白高度磷酸化及AD的可能的蛋白質激酶。
在本研究中，我們主要以免疫螢光分析（immunofluorescence analysis），免疫沈澱法（co-immunoprecipitation）及GST融合蛋白pull down assay等方法來探討Tau及其mutants與tubulin的subcellularlocalization和交互作用，結果發現full length Tau（Tau WT)， Tau的N端（Tau-N），Tau的C端（Tau-C）皆會與tubulin結合，但是Tau121-244則表現於細胞核內。並進一步以tubulin聚合分析（tubulin assembly assay）來探討Tau或Tau mutants是否具有促進tubulin 聚合的能力，結果發現只有Tau WT具有促進tubulin聚合的能力，而Tau-N或Tau-C雖可以in vivo及in vitro情況下與tubulin相結合，但卻失去了促進tubulin聚合的能力，我們的結論是，Tau WT在促使tubulin 聚合時，需同時具有N端及C端的domains。在另一Tau的調控機制方面，我們以不同蛋白激酶質進行磷酸化分析及並利用定點突變方法，驗證了cdc2與GSK3β皆會磷酸化Tau於T231的位置，且T231對上述之蛋白質激酶而言為重要的磷酸化位點。最後並以tubulin 聚合分析證明GSK3β磷酸化會抑制Tau蛋白之促進tubulin聚合的能力，並推測Tau蛋白T231位點之磷酸化，可能具有調控Tau蛋白之功能。

Tau protein is one of the microtubule-associated proteins (MAPs) and mainly expressed in neuronal cells. It hasbeen demonstrated that Tau may play an important role in regulating microtubule dynamic in neurons. Structurally and functionally, Tau protein composed of regulatory projection domain in N-terninus and microtubule-binding domain in C-terminus. It has been shown that the biological function of Tau protein was regulated by phosphorylation and dephosphorylation. In Alzheimer’s disease (AD) brain, hyperphosphorylated Tau caused by over active kinases may contribute to the disassociation of Tau from microtubule and form the pathologically hallmarker, paired helical filaments (PHFs). The reason to study cdc2 and GSK3β is two folds. First, both cdc2 and GSK3β activities are raised abbrently in AD brain. Second, the phosphorylation sites of cdc2 and GSK3β have been identified as those in PHFs.These prompted us to regard cdc2 and GSK3β as candidates that hyperphosphorylated Tau in AD.
In the following study, we used immunofluorescence analysis, co-immunoprecipitation and GST-fusion protein pull down assay to clarify the subcellular localization of Tau. We also shown that the interaction between tubulin withfull length Tau (Tau WT) and some Tau mutants that we found that not only Tau WT, but also N-terminus of Tau (Tau-N) and C-terminus of Tau (Tau-C) can bind to tubulin. Surprisingly, we observed that a fragment of N-terminus, Tau 122-244, localized in nucleus. Furthermore, we used tubulin assembly assay to test if tau or its mutants can promote tubulin assembly in vitro. Results showed that only Tau WT can promote tubulin assembly in vitro but not Tau-N or Tau-C. Although Tau-N or Tau-C can bind to tubulin in vivo and in vitro, these mutants did not remain the ability to promote tubulin assembly that suggested both functional domains, N-terminus and C-terminus of Tau, are necessary and essential for the biological function of Tau. On the other hand, we used of phosphorylation assay and site directed mutagenesis to demonstrate that T231 of Tau is one of important phosphorylation sites of cdc2 and GSK3β. Finally, we used tubulin assembly assay to show that phosphorylated Tau by GSK3β can negatively regulate the ability of Tau to promote tubulin assembly that indicated that the phosphorylation at T231 may play a role in regulating Tau.

一、中文摘要…………………………………………………… …...i
二、英文摘要………………………………………………………....iii
三、英文縮寫表………………………………………………………..v
四、序言……………………………………………………………….1
五、材料方法………………………………………………………….9
六、結果……………………………………………………………...24
七、討論……………………………………………………………...33
八、參考文獻………………………………………………………...37
九、圖………………………………………………………………...42
十、附錄……………………………………………………………...61

Buee L., Bussiere T., Buee-Scherrer, V., Delacourte, A., and Hof, P. R. (2000). Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res. Brain Res. Rev. 33:95-130.
2. Spillantini, M. G., and Goedert, M. (1998). Tau protein pathology in neurodegenerative diseases. Trends Neurosci. 21:428-33.
3. Trojanowski, J. Q., Shin, R. W., Schmidt, M. L., and Lee, V. M. (1995). Relationship between plaques, tangles, and dystrophic processes in Alzheimer's disease. Neurobiol. Aging 16:335-40.
4. Billingsley, M. L., and Kincaid, R. L. (1997). Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration. Biochem. J. 323:577-91.
5. Lee, V. M., Balin, B. J., Otvos, L. Jr., and Trojanowski, J. Q. (1991). A68: a major subunit of paired helical filaments and derivatized forms of normal Tau. Science 251:675-8.
6. Roland, Brandt. (1996). The tau proteins in neuronal growth and development. Frontiers. in Bioscience 1:118-130.
7. Iqbal, K., Zaidi, T., Bancher, C., and Grundke-Iqbal, I. (1994). Alzheimer paired helical filaments. Restoration of the biological activity by dephosphorylation. FEBS Lett. 349:104-8.
8. Brandt, R. (2001). Cytoskeletal mechanisms of neuronal degeneration. Cell Tissue Res. 305:255-65.
38 9. Hagiwara, H., Yorifuji, H., Sato-Yoshitake, R., and Hirokawa, N. (1994). Competition between motor molecules (kinesin and cytoplasmic dynein) and fibrous microtubule-associated proteins in binding to microtubules. J. Biol. Chem. 269:3581-9.
10. Mandelkow, E., Song, Y. H., Schweers, O., Marx, A., and Mandelkow, E. M. (1995). On the structure of microtubules, tau, and paired helical filaments. Neurobiol. Aging 16:347-54.
11. Alonso, A. D., Zaidi, T., Novak, M., Barra, H. S., and Grundke-Iqbal, K. (2001). Interaction of tau isoforms with Alzheimer's disease abnormally hyperphosphorylated tau and in vitro phosphorylation into the disease-like protein. J. Biol. Chem. 276:37967-73.
12. Tseng, H. C., Lu, Q., Henderson, E., and Graves, D. J. (1999). Phosphorylated tau can promote tubulin assembly. Proc. Natl. Acad. Sci. (U.S.A.) 96:9503-8.
13. Schweers, O., Schonbrunn-Hanebeck, E., Marx, A., and Mandelkow. E. (1994). Structural studies of tau protein and Alzheimer paired helical filaments show no evidence for beta-structure. J. Biol. Chem. 269:24290-7.
14. Goode, B. L., Chau, M., Denis, P. E., and Feinstein, S. C. (2000).Structural and functional differences between 3-repeat and 4-repeat tau isoforms.Implications for normal tau function and the onset of neurodegenetative disease. J. Biol. Chem. 275:38182-9.
15. Brandt, R., Leger, J., and Lee, G. (1995). Interaction of tau with the
39 neural plasma membrane mediated by tau's amino-terminal projection domain. J. Cell Biol. 131:1327-40.
16. Husseman, J.W., Nochlin, D., and Vincent, I. (2000). Mitotic activation: a convergent mechanism for a cohort of neurodegenerative diseases. Neurobiol. Aging 21:815-28.
17. Ishiguro, K., Shiratsuchi, A., Sato, S., Omori, A., Arioka, M., Kobayashi. S., Uchida, T., and Imahori, K. (1993). Glycogen synthase kinase 3 beta is identical to tau protein kinase I generating several epitopes of paired helical filaments. FEBS Lett. 325:167-72.
18. Imahori, K., Hoshi, M., Ishiguro, K., Sato, K., Takahashi, M., Shiurba, R., Yamaguchi, H., Takashima, A., and Uchida, T. (1998). Possible role of tau protein kinases in pathogenesis of Alzheimer's disease. Neurobiol. Aging 19:S93-8.
19. Preuss, U., and Mandelkow, E. M. (1998). Mitotic phosphorylation of tau protein in neuronal cell lines resembles phosphorylation in Alzheimer's disease. Eur. J. Cell Biol. 76:176-84.
20. Paudel, H. K. (1997). Phosphorylation by neuronal cdc2-like protein kinase promotes dimerization of Tau protein in vitro. J. Biol. Chem. 272:28328-34.
21. Lucas, J. J., Hernandez, F., Gomez-Ramos, P., Moran, M. A., Hen, R., and Avila, J. (2001). Decreased nuclear beta-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3beta conditional transgenic mice. EMBO J. 20:27-39.
40 22. Patrick, G. N., Zukerberg, L., Nikolic, M., de la Monte, S., Dikkes, P., and Tsai, L. H. (1999). Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature 402:615-22.
23. Grimes, C. A., and Jope, R. S. (2001). The multifaceted roles of glycogen synthase kinase 3beta in cellular signaling. Prog. Neurobiol. 65:391-426.
24. Ali, A., Hoeflich, K. P., Woodgett, J. R. (2001). Glycogen synthase kinase-3: properties, functions, and regulation. Chem. Rev. 101:2527-40.
25. Kaytor, M. D, and Orr, H. T. (2002). The GSK3beta signaling cascade and neurodegenerative disease. Curr. Opin. Neurobiol. 12:275-8.
26. Lu, P. J., Wulf, G., Zhou, X. Z., Davies, P., and Lu, K. P. (1999). The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein. Nature 399:784-8.
27. Jicha, G. A., Lane, E., Vincent, I., Otvos, L. Jr., Hoffmann, R., and Davies, P. (1997). A conformation- and phosphorylation-dependent antibody recognizing the paired helical filaments of Alzheimer's disease. J. Neurochem. 69:2087-95.
28. Vincent, I., Rosado, M., and Davies, P. (1996). Mitotic mechanisms in Alzheimer's disease? J. Cell Biol. 132:413-25.
29. Daly, N. L., Hoffmann, R., Otvos, L. Jr., and Craik, D. J. (2000). Role of phosphorylation in the conformation of tau peptides implicated in Alzheimer's disease. Biochemistry 39:9039-46.
41 30. Loomis, P. A., Howard, T. H., Castleberry, R. P., and Binder, L. I. (1990). Identification of nuclear tau isoforms in human neuroblastoma cells. Proc. Natl. Acad. Sci. (U. S. A.) 87:8422-6.
31. Sang, H., Lu, Z., Li, Y., Ru, B., Wang, W., and Chen, J. (2001). Phosphorylation of tau by glycogen synthase kinase 3beta in intact mammalian cells influences the stability of microtubules. Neurosci. Lett. 312:141-4.
32. Sun, W., Qureshi, H. Y., Cafferty, P. W., Sobue, K., Agarwal-Mawal, A., Neufield, K. D., and Paudel, H. K. (2002). Glycogen synthase kinase-3beta is complexed with tau protein in brain microtubules. J. Biol. Chem. 277:11933-40.
33. Brand, R., Lee, G., Teplow, D.B., Shalloway, D., and Abdel-Ghany, M. (1994). Differential effect of phosphorylation and substrate modulation on tau's ability to promote microtubule growth and nucleation. J. Biol. Chem. 269:11776-82.