進階搜尋


 
系統識別號 U0026-0812200911564207
論文名稱(中文) 探討MST3調控細胞移動的機轉
論文名稱(英文) Study on the Mechanism of Cell Motility Mediated by MST3
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 94
學期 2
出版年 95
研究生(中文) 謝宛蓉
研究生(英文) Wan-Jung Hsieh
電子信箱 ymmt93picture@yahoo.com.tw
學號 s1693405
學位類別 碩士
語文別 中文
論文頁數 98頁
口試委員 口試委員-劉校生
口試委員-呂增宏
指導教授-賴明德
中文關鍵字 細胞爬行  細胞移動 
英文關鍵字 MST3  migration 
學科別分類
中文摘要 細胞的移動在生物體內胚胎發育以及許多生理功能性上,扮演必須且重要的過程。Mammalian Sterile 20-like kinase 3 (MST3) 歸屬於STE20 serine/threonine kinase家族,其生物功能由我們實驗室發現與細胞移動有關。我們利用了RNAi技術抑制了爬行較慢MCF7細胞株的內生MST3,發現了其功能可抑制細胞伸出突觸及爬行,而利用帶有靜突變的MST3則可回復RNAi細胞株爬行較快的現象;或是利用大量表現野生型與突變其自體磷酸化位置T178A突變型的MST3,也可觀察到MST3抑制爬行較快的MDCK細胞株爬行,而T178A-MST3則完全喪失其抑制爬行的能力。我們同時也探討MST3調控細胞爬行所牽涉的機轉。在將MST3抑制後,我們觀察到paxillin的tyrosine 31與118的位置磷酸化程度下降;且外送回野生型的MST3到爬行快速的MDCK細胞株後,發現paxillin磷酸化程度上升,並與細胞爬行速度有相關性。但由於MST3為serine/threonine kinase,應無法直接影響paxillin tyrosine磷酸化,因此MST3應該是影響另一個與paxillin相關的蛋白,進而調控paxillin磷酸化程度。在這裡我們推測Protein tyrosine phosphatase-PEST,其為一個已知會與paxillin結合的酪胺酸去磷酸酶。在我們研究中,我們發現recombinant MST3可以直接磷酸化PTP-PEST peptide。在MDCK細胞中大量表現MST3可抑制PTP-PEST酵素活性,而在MST3-RNAi MCF7細
胞株中,其PTP-PEST酵素活性也因MST3被抑制而隨之上升。因此我們推測MST3藉由磷酸化PTP-PEST,使其酵素活性降低,進而無法將paxillin去磷酸化,維持著paxillin Y31與Y118高度磷酸化,而抑制了細胞的爬行。第二部份,MST3不尋常地利用錳離子當酵素活性的輔因子,與一般serine/threonine kinase喜好鎂離子不同。我們經由預測比對可能調控MST3喜好錳離子的位置,選擇Q47位置突變成E。此突變株在錳離子存在下酵素活性降低,而在送入MDCK細胞中後,發現此突變株喪失了部分抑制爬行的能力,也改變了paxillin磷酸化的程度。因此MST3如何藉由以二價陽離子錳當輔因子來調控paxillin之磷酸化,而控制細胞爬行的內部分子機制,是未來更進一步研究的方向。
英文摘要 Cell motility is an important process that is essential for embryonic development and life-long physiological processes. Mammalian Sterile 20-like kinase 3 (MST3) belongs to the STE20 serine/threonine protein kinase family, and its biological functions in cell motility had been demonstrated by our laboratory. Either overexpression or down-regulation of MST3 affects cellular protrusion and migration. In MST3-RNAi MCF7 cells, the alteration of cellular migration and protruding can be rescued by RNAi-resistant MST3. We also investigate the molecular mechanism how MST3 regulates cell migration. We observed that phosphorylation on Tyrosine-118 and 31 of paxillin was decreased by MST3 RNAi expression, and the extent of pY31 and pY118 of paxillin is correlated with migration behavior of MDCK cells expressing various forms of MST3. But MST3 is a kinase involved in phosphorylation at serine or threonine residue, and is unlikely to directly change the phosphorylation of paxillin. MST3 may affect the phosphorylation of paxillin through other paxillin-interacting proteins. We hypothesized that PTP-PEST, a protein tyrosine phosphatase associating with paxillin, may be the direct target of MST3. In our study, recombinant MST3 could phosphorylate the peptide of PTP-PEST in vitro. Overexpression of MST3 in MDCK cells can inhibit PTP-PEST phosphatase activity, and the activity of PTP-PEST in MST3-RNAi MCF7 cells was enhanced. Therefore we suggest MST3 regulates cellular migration with alteration of paxillin phosphorylation through inhibiting PTP-PEST activity. Secondly, a unique feature for MST3 is
the preference for Mn2+ as the cofactor in kinase reaction, which is unusual for a serine/threonine kinase. We predicted the possible residues involved in cofactor preference and selected Q47 as our potential targets. The Q47E mutant showed reduced kinase activity in the presence of Mn2+ in vitro and reduced inhibition of migration ability comparing with wild type MST3. The detailed mechanism of metal ion cofactor and migration awaits further investigation.
論文目次 緒論
一、Ste20 (sterile 20)族群蛋白激酶             1
二、MST家族蛋白的功能                    2
三、細胞爬行移動及其機轉                  3
四、金屬離子在激酶所扮演的角色               6
五、研究目標與策略                     7

材料與方法
一、細胞培養與藥物處理                   9
二、質體製備                        14
三、構築pHA-mouse-wt-ATF6、pHA -mouse-N’-ATF6
、353-Grp78-Luc-reporter gene                19
四、基因殖入轉染                      27
五、西方墨點法                        28
六、免疫組織化學染色法                  35
七、報導基因分析                   37
八、流式細胞儀分選細胞                  38
九、細胞移動分析                   38
十、細胞癒合分析                   40
十一、免疫沉澱法                41
十二、 磷酸酶酵素能力試驗             43
十三、 去磷酸酶酵素能力試驗                 45
十四、 動物實驗與治療效果評估方式           46


結果
一、降低細胞內MST3表現可促進細胞爬行能力      48
二、外送MST3進入RNAi clone可回復其抑制細胞爬行的能力  49
三、過度表現MST3可抑制快速爬行細胞株MDCK的移動能力     50
四、MST3抑制細胞爬行是經由維持paxillin之磷酸化所造成    51
五、Recombinant MST3可直接磷酸化PTP-PEST peptide   52
六、細胞內過度表現MST3可抑制PTP-PEST去磷酸酶活性  52
七、改變MST3對錳離子的喜好與其調控細胞爬行的能力有關   53

結論      55
討論      56
參考文獻         64
圖       70
附圖緒論         82
附圖      89
自述      98
參考文獻 Angers-Loustau, A., Cote, J.F., Charest, A., Dowbenko, D., Spencer, S., Lasky, L.A., and Tremblay, M.L. Protein tyrosine phosphatase-PEST regulates focal adhesion disassembly, migration, and cytokinesis in fibroblasts. J. Cell Biol. 144:1019-1031, 1999.

Chan, E.H., Nousiainen, M., Chalamalasetty, R.B., Schafer, A., Nigg, E.A. and Sillje, H.H. The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1. Oncogene. 24:2076-2086, 2005.

Chebassier, N., El Houssein, O., Viegas, I. and Dreno, B.: In vitro induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 expression in keratinocytes by boron and manganese. Exp Dermatol. 13:484-490, 2004.

Creasy, C.L., Ambrose, D.M. and Chernoff, J.: The Ste20-like protein kinase, Mst1, dimierizes and contains inhibitory domain. J. Biol. Chem. 271:21049-21053, 1996.

Dan, I., Watanabe, N.M. and Kusumi, A.: The Ste20 group kinases as regulators of MAP kinase cascades. Trends Cell Biol. 11:220-230, 2001.

Edwards, J.G., Hamedde, H. and Campbell, G. Induction of fibroblaset spreading by Mn2+: a possible role for unusual binding sites for divalent cations in receptors for proteins containing Arg-Gly-Asp. J. Cell Sci. 89:507-513, 1988.

Garton, A. and Tonks, N.: PTP-PEST: a protein tyrosine phosphatase regulated by serine phosphorylation. EMBO. J. 13:3763-3771, 1994.

Garton, A.J. and Tonis, N.K. Regulation of fibroblast motility by the protein tyrosine phosphatase PTP-PEST. J. Biol. Chem. 274:3811-3818, 1999.

Glantschnig, H., Rodan, G.A. and Reszka, A.A. Mapping of MST1 kinase sites of phosphorylation, ctivation and autophosphorylation. J. Biol. Chem. 277:42987-42996, 2002.

Graves, J.D., Gotoh, Y., Draves, K.E., Ambrose, D., Han, D.K., Wright, M., 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.

He. H., McColl. K. and Distelhorst. C.W.: Involvement of c-Fos in signaling grp78 induction following ER calcium release. Oncogene. 19:5936-5943, 2000.

Hong. M., Luo. S., Baumeister. P., Huang. J.M., Gogia. R.K., Li. M. and Lee. A.S.: Underglycosylation of ATF6 as a novel sensing mehanism for activation of the unfolded protein reponse. J. Biol. Chem. 279:11354-11363, 2004.

Horwitz, R. and Web, D. Cell migration. Curr Biol. 13:R756-759, 2003.

Huang, C.Y., Wu, Y.M., Hsu, C.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., Yuan, C.J.: Caspase activation of mammalian sterile 20-like kinase 3 (MST3). J. Biol. Chem. 277:34367-34374, 2002.

Iwasaki, T., Nakata, A., Mukai, M., Shinkai, K., Yano, H., Sabe, H., Schaefer, E., Tatsuta, M., Tsujimura, T., Terada, N., Kakishita, E. and Akedo, H.: Involvement of phosphorylation of Tyr-31 and Tyr-118 of paxillin in MM1 cancer cell migration. Int. J. Cancer. 97:330-335, 2002.

Jamieson, J.S., Tumbarello, D.A., Halle, M., Brown, M.C., Tremblay, M.L., and Turner, C.E.: Paxillin is essential for PTP-PEST-dependent regulation of cell spreading and motility: a role for paxillin kinase linker. J. Cell Sci. 118:5835-5847, 2005.

Leberer. E., Dignard, D., Harcus, D., Tomas, D.Y. and Whiteway, M. The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signaling components. EMBO J. 11:4815-4824, 1992.

Lee, K.K., Ohyama, T., Yajima, N., Tsubuki, S. and Yonehara, S.: MST, a physiological caspase substrate, highly sensitizes apoptosis both upstream and downstream of caspase activation. J. Biol. Chem. 276:19276-19285, 2001.

Lee, K.K. and Yonehara, S. Phosphorylation and dimerization regulate nucleocytoplasmic shuttling of mammalian STE20-like kinase (MST). J. Biol. Chem. 277:12351-12358, 2002.

Lee, W.S., Hsu, C.Y., Wang, P.L., Huang, C.Y., Chang, C.H. and Yuan, C.J. Identification and characterization of the nuclear import and export signals of the mammalian Ste20-like protein kinase 3. FEBS Lett. 572:41-45, 2004.

Lin, J.L., Chen, H.C., Fang, H.I., Robinson, D., Kung, H.J. and Shih, H.M.: MST4, a new Ste20-related kinase that mediates cell growth and transformation via modulating ERK pathway. Oncogene. 20:6559-6569, 2001.

Liu, F., Hill, D.E. and Cheronff J.: Direct binding of the praline-rich region of protein tyrosine phosphatase 1B to the Src homology 3 domain of p130(Cas). J. Biol. Chem. 271:31290-31295, 1996.

Lu, T.J., Huang, C.Y., Yuan, C.J., Lee, Y.C., Leu, T.H., Chang, W.C., Lu, T.L., Jeng, W.Y. and Lai, M.D.: Zinc ion acts as a cofactor for serine/threonine kinase MST3 and has a distinct role in autophosphorylation of MST3. J. Inorg. Biochem. 99:1306-1313, 2005.

Ly, D.P., Zazzali, K.M. and Corbett, S.A.: De novo expression of the integrin alpha5beta1 regulates alphavbeta3-mediated adhesion and migration on fibrinogen. J. Biol. Chem. 278:21878-21885, 2003.

Nakamura, K., Yano, H., Uchida, H., Hashimoto, S., Schaefer, E. and Sabe, H.: Tyrosine phosphorylation of paxillin alpha is involved in temporospatial regulation of paxillin-containing focal adhesion formation and F-actin organization in motile cells. J. Biol. Chem. 275:27155-27164, 2000.

O’Neill, E., Rushworth, L., Baccarini, M. and Kolch, W. Role of the kinase MST2 in suppression of apoptosis by the proto-oncogene product Raf-1. Science. 306:2267-2270, 2004.

Osada, S., Izawa, M., Saito, R., Mizuno, K., Suzuki, A., Hirai, S., Ohno, S.: YSK1, a novel mammalian protein kinase structurally related to Ste20 and SPS1, but is not involved in the known MAPK pathways. Oncogene. 14:2047-2057, 1997.

Paulsson, K. and Wang, P.: Chaperones and folding of MHC class I molecules in the endoplasmic reticulum. Biochimica et Biophysica Acta. 1641:1-12, 2003.

Petit, V., Boyer, B., Lentz, D., Turner, C.E., Thiery, J.P., and Valles, A.M.: Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells. J. Cell Biol. 148:957-970, 2000.

Pombo, C.M., Bonventre, J.V., Molnar, A., Kyriakis, J. and Force, T. Activation of a human Ste20-like kinase by oxidant stress defines a novel stress reponse pathway. EMBO J. 15:4537-4546, 1996.

Preisinger, C., Short, B., De Corte, V., Bruyneel, E., Haas, A., Kopajtich, R., Gettemans, J., Barr, F.A.: YSK1 is activated by the Golgi matrix protein GM130 and plays a role in cell migration through its substrate 14-3-3zeta. J. Cell Biol. 164:1009-1020, 2004.

Qian, Z., Lin, C., Espinosa, R., LeBeau, M. and Rosner, M.R. Cloning and characterization of MST4, a novel Ste20-like kinase. J. Biol. Chem. 276:22439-22445, 2001.

Reiter, T.A., Reiter, N.J. and Rusnak, F. Mn2+ is a notive metal ion activator for bacteriophage lambda protein phosphatase. Biochemistry. 41:15404-15409, 2002.

Ridley, A.J., Schwartz, M.A., Burridge, K., Firtel, R.A., Ginsberg, M.H., Borisy, G., Parsons, J.T. and Horwitz, A.R. Cell migration: integrating signals form front to back. Science. 302:1704-1709, 2003.


Romanova, L.Y., Hashimoto, S., Chay, K.O., Blagodklonny, M.V., Sabe, H. and Mushinski, J.F. Phosphorylation of paxillin tyrosines 31 and 118 controls polarization and motility of lymphoid cells and is PMA-sensitive. J. Cell Sci. 117:3759-3768, 2004.

Sastry, S.K., Lyons, P.D., Schaller, M.D. and Burridge, K.: PTP-PEST controls motility through regulation of Rac1. J. Cell Biol. 115:4305-4316, 2002.

Schaller, M.D.: FAK and paxillin: regulators of N-cadherin adhesion and inhibitors of cell migration? J. Cell Biol. 166:157-159, 2004.

Schinkmann, K. and Blenis, J.: Cloning and characterization of a human STE20-like protein kinase with unusual cofactor requirements. J. Biol. Chem. 272:28695-28703, 1997.

Stegert, M.R., Hergovich, A., Tamaskovic, R., Bichsel, S.J., Hemmings, B.A.: Regulaiton of NDR protein kinase by hydrophobic motif phosphorylation mediated by the mammalian Ste20-like kinase MST3. Mol. Cell Biol. 25:11019-11029, 2005.

Sung, V., Luo, W., Qian, D., Lee, I., Jallal, B. and Gishizky, M.: The Ste20 kinase MST4 plays a role in prostate cancer progression. Cancer Res. 63:3356-3363, 2003.

Takino, T., Tamura, M., Miyamori, H., Araki, M., Matsumoto, K., Sato, H., and Yamada, K.M.: Tyrosine phosphorylation of the CrkII adaptor protein modulates cell migration. J. Cell Sci. 116:3145-3155, 2003.

Tamaskovic, R., Bichsel, S.J. and Hemmings, B.A. NDR family of AGC kinases—essential regulators of the cell cycle and morphogenesis. FEBS Lett. 546:73-80, 2003.

Tsubouchi, A., Sakakura, J., Yagi, R., Mazaki, Y., Schaefer, E., Yano, H. and Sabe, H.: Localized suppression of RhoA activity by Tyr31/Tyr118 phosphorylated paxillin in cell adhesion and migration. J. Cell Biol. 159:673-683, 2002.

Turner, C.E.: Paxillin and focal adhesion signaling. Nat Cell Biol. 2:E231-E236, 2000

Webb, D.J., Parsons, J.T. and Horwitz, A.F. Adhesion assembly, disassembly and turnover in migrating cells—over and over and over again. Nat Cell Biol. 4:E97-100, 2002.

Yano, H., Uchida, H., Iwasaki, T., Mukai, M., Akedo, H., Nakamura, K., Hashimoto, S., and Sabe, H.: Paxillin alpha and Crk-associated substrate exert opposing effects on cell migration and contact inhibition of growth through tyrosine phosphorylation. Proc Natl Acad Sci U S A. 97:9076-9081, 2000

Yano, H., Mazaki, Y., Kurokawa, K., Hanks, S.K., Matsuda, M., and Sabe, H.: Roles played by a subset of integrin signaling molecules in cadherin-based cell-cell adhesion. J. Cell Biol. 166:283-295, 2004.

Zhou, T.H., Ling, K., Guo, J., Zhou, H., Wu, Y.L., Jing, Q., Ma, L. and Pei, G. Identification of a human brain-specific isoform of mammalian STE20-like kinase 3 that is regulated by cAMP-dependent protein kinase. J. Biol. Bhem. 275:2513-2519, 2000.

賴文揚,探討MST3激酶對於細胞型態及移動所執行的功能。
國立成功大學生物化學研究所碩士論文,2004。

趙詠梅,探討在內質網壓力下肝細胞中的c-Jun所扮演的可能角色。
國立成功大學生物化學暨分子生物研究所碩士論文,2005。

陸德容,MST3之功能為抑制細胞爬行且此功能受其自體磷酸化及離子使用特性所影響。
國立成功大學基礎醫學研究所博士論文,2006。
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2007-07-13起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2007-07-13起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw