臺灣博碩士論文加值系統

一型蛋白質磷酸水解酶（PP1）為重要的絲胺酸/酥胺酸蛋白質磷酸水解酶，在體內PP1會和調控次單位結合，調控次單位可影響PP1於細胞內的分布並藉由反轉蛋白質激酶反應調節PP1對專一受質的活性。Phostensin (protein phosphatase 1 F-actin cytoskeleton targeting subunit) 是由KIAA1949所轉譯的蛋白質，包含165 個胺基酸，肌動蛋白動態分析顯示phostensin可以降低F-型肌動蛋白去聚合作用的速率，被gelsolin結合在barbed end的F-型肌動蛋白並不影響phostensin對於肌動蛋白動態的調節能力，測量thioredoxin-phostensin抑制Gelsolin-capped F-型肌動蛋白pointed ends聚合作用速率的能力得知thioredoxin-phostensin對F-型肌動蛋白pointed ends的結合活性，thioredoxin-phostensin對F-型肌動蛋白pointed ends的解離平衡常數(Kd)為1.1 ± 0.5μM。研究結果指出phostensin可藉由影響F-型肌動蛋白pointed ends的聚合及去聚合作用而調節肌動蛋白動力平衡。

Protein phosphatase 1 (PP1), a major serine/threonine protein phosphatase, is associated in vivo with a family of regulatory subunits that target the enzyme to different subcellular locations and modulate the activity of the enzyme toward specific substrates at these sites by reversing the action of protein kinases. Phostensin, a protein phosphatase 1 F-actin cytoskeleton targeting subunit encoded by KIAA1949, consists of 165 amino acids. Actin dynamics assay shows that phostensin decreased the depolymerization rates of actin filaments. Capping the barbed ends of actin filaments by gelsolin did not affect phostensin on modulation of actin dynamics. The pointed end-capping activities of thioredoxin-phostensin were measured their ability to inhibit the initial rates of actin elongation from the pointed ends of gelsolin-capped actin filaments. The equilibrium dissociation constant (Kd) for thioredoxin-phostensin was 1.1 ± 0.5μM. Taken together, our results suggested that phostensin is an actin filament pointed end-capping protein and is capable of modulating actin dynamics.

中文摘要..................................................................................................i
英文摘要.................................................................................................ii
目錄........................................................................................................iii
圖目錄..................................................................................................v
一、 序論..................................................................................................1
1-1背景介紹.....................................................................................1
1-2研究動機.....................................................................................9
二、 實驗材料與方法............................................................................11
2-1緩衝溶液配製...........................................................................11
2-2質體構築(plasmid construction) ..............................................13
2-3蛋白質………………………...................................................15
2-4 西方墨點法(western blot)……………………………………20
2-5肌動蛋白動力學分析(actin dynamics assay) ..........................21
三、 實驗結果........................................................................................23
3-1抗phostensin單株抗體............................................................23
3-2 Phostensin會抑制F-型肌動蛋白（—）端的去聚合作用….25
四、 討論................................................................................................28
五、 參考文獻........................................................................................31
附圖.............................................................................................35
附錄........................................................................................................48

1.Cohen, P. (1989) The structure and regulation of protein phosphatases. Annu. Rev. Biochem. 58, 453-508
2.Shenolinkar, S., and Nairn, A. C. (1991) Protein phosphatases: Recent Progress. In Greengard, P. and Robinson, G.. A. (eds.) , Advances in Second Messengers and Phosphoprotein Research. Raven Press, New York, 23, 1-121
3.Shenolinkar, S., and Ingebritsen, T. S. (1984) Protein (serine and threonine) phosphate phosphatases. Methods In Enzymology 107, 102-129
4.Bordelon, J. R., Smith Y., Nairn, A. C., Colbran, R. J., Greengard, P., and Muly, E. C. (2005) Differential localization of protein phosphatase-1? β, and γ1 isoforms in primate prefornal cortex. Cereb. Cortex. Mar 9
5.Faux, M. C., and Scott, J. D. (1996) More on target with protein phosphorylation: conferring specificity by location. Trends In Biochemical Sciences 21 (8) 312-315
6.Zhao, S., Lee, E. Y. C. (1997) A protein phosphatase-1-binding motif identified by the panning of a random peptide display library J. Biol. Chem. 272, 28368-28372
7.Satoh, A., Nakanishi, H., Obasishi, H., Wada, M., Takahashi, K., Satoh, K. Nishioka, H., Matsuura, Y., Mizoguchi, A., and Takai, Y. (1998) Neurabin-II/spinophilin. An actin filament-binding protein with one PDZ domain localized at cadherin-based cell-cell adhesion sites. J. Biol. Chem. 273, 3470-3475
8.Allen, P. B., Ouimet, C. C., and Grengard, P. (1997) Spinophilin, a novel protein phosphatase 1 binding protein localized to dendritic spines. Proc. Natl. Acad. Sci USA 94, 9956-9961
9.Terry-Lorenzo, R. T., Elliot, E., Weiser, D. C., Prickett, T. D. Brautigan, D. L., Nairn, A. C., and Greengard, P. (2002) Neurabins Recruit Protein Phosphatase-1 and Inhibitor-2 to the Actin Cytoskeleton. J. Biol. Chem. 277, 46535-46543
10.Dos remedios,C. G., Chhabra,D., Kekic, M., Dedova, I. V., Tsubakihara, M., Berry, D. A., and Nosworthy, N. J. (2003) Actin Binding Proteins: Regulation of Cytoskeletal Microfilaments. Physiol. Rev. 83, 433-473
11.De, L. A., Cruz, E. M., and Pollard, T. D. (1995) Nucleotide-free actin: stabilization by sucrose and nucleotide binding kinetics. Biochemistry 34, 5452–5461
12.Strzelecka-Golaszwaska, H. (2001) Divalent cations, nucleotides and actin structure. Res. Prob. Cell Differ. 32, 23-42
13.Schutt, C. E., Myslik, J.C., Rozycki, M. D., Gooneskere, N., and Lindverg, U. (1993) The structure of crystalline profilin-beta-actin. Nature 365, 810- 816
14.Cooper, J. A., Giardini, P. A., Soo, F. S., and Theriot, J. A. (2000) Secrets of actin-based motility revealed by a bacterial pathogen. Nature Rev. Mol. Cell Biol. 1, 110-119
15.Pollard, T. D. (1986) Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J Cell Biol 103, 2747–2754
16.Wegner, A. (1976) Head to tail polymerization of actin. J. Mol. Biol. 108, 139-150
17.Pollard, T. D., and Cooper, J. A. (1986) Actin and actin-binding proteins: a critical evaluation of mechanisms and function. Annu. Rev. Biochem. 55, 987–1035
18.Didry, D., Carlier, M.F., and Pantaloni, D. (1998) Synergy between actin depolymerizing factor cofilin and profilin in increasing actin filament turnover. J. Biol. Chem. 273, 25602-25611
19.Kao, S.C., Chen, C.Y., Wang, S.L., Yang, J.J., Hung, W.C., Chen, Y.C., Lai, N.S., Liu, H.T., Huang, H.L., Chen, H.C., Lin, T.H., and Huang, H.B. (2007) Identification of phostensin, a PP1 F-actin cytoskeleton targeting subunit. Biochem. and Biophys. Res. Commun. 356, 594-598.
20.Shigenari, A., Ando, A., Renard, C., Chardon, P., Shiina, T., Kulski, J. K., Yasue, H., and Inoko, H. (2004) Nucleotide sequencing analysis of the swine 433-kb genomic segment located between the non-classical and classical SLA class I gene clusters. Immunogenetics 55, 695-705.
21.Weber, A., Pennise, C.R., Babcock, G.G. and Fowler, V.M. (1994) Tropomodulin caps the pointed ends of actin filaments. J. Cell Biol. 127, 1627-1635.
22.Weber, A., Pennise, C.R. and Fowler, V. (1999) Tropomodulin increases the critical concentration of barbed end-capped actin filaments by converting ADP•Pi-actin to ADP-actin at all pointed filament ends. J .Biol. Chem. 274, 34637-34645.
23.Flowler, V.M., Greenfield, N.J. and Moyer, J. (2003) Tropomodulin contains two actin filament pointed end-capping domain. J. Biol. Chem. 278, 40000-40009.
24.Nakanishi, H., Obaishi, H., Satoh, A., Wada, M., Mandai, K., Satoh, K., Nishioka, H., Matsuura, Y., Mizoguchi, A. and Takai, Y. (1997) Neurabin: a novel neural tissue-specific actin filament-binding protein involved in neurite formation. J. Cell Biol. 139, 951-961.
25.Terry-Lorenzo, R.T., Carmody, L.C., Voltz, J.W., Connor, J.H., Li, S., Smith, F.D., Milgram, S.L., Colbran, R.J. and Shenolikar, S. (2002) The neuronal actin-binding proteins, neurabin I and neurabin II, recruit specific isoforms of protein phosphatase-1 catalytic subunits. J. Biol. Chem. 277, 27716-27724.
26.Oliver, C.J., Terry-Lorenzo, R.T., Elliott, E., Bloomer, W.C., Li, S., Brautigan, D.L., Colbran, R.J. and Shenolikar, S. (2002) Targeting protein phosphatase 1 (PP1) to the actin cytoskeleton: the neurabin I/PP1 complex regulates cell morphology. Mol. Cell Biol. 22, 4690-4701
27.Terry-Lorenzo, R.T., Roadcap, D.W., Otsuka, T., Blanpied, T.A., Zamorano, P.L., Garner, C.C., Shenolikar, S. and Ehlers, M.D. (2005) Neurabin/protein phosphatase-1 complex regulates dendritic spine morphogenesis and maturation. Mol. Biol. Cell 16, 2349-2362.
28.Cohen, P.T.W. (2002) Protein phosphatase 1-targeted in many directions. J .Cell Sci. 15, 241-256.