進階搜尋


 
系統識別號 U0026-0812200911033028
論文名稱(中文) 蛋白質去磷酸化在表皮生長因子誘導人類上皮癌A431細胞中c-Jun及Sp1交互作用所參與的角色
論文名稱(英文) Involvement of protein dephosphorylation in EGF-induced interaction between c-Jun and Sp1 in A431 cells
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 92
學期 1
出版年 93
研究生(中文) 陳韻如
研究生(英文) Yun-Ju Chen
電子信箱 crita0326@yahoo.com.tw
學號 s2690407
學位類別 碩士
語文別 中文
論文頁數 69頁
口試委員 口試委員-呂增宏
指導教授-張文昌
口試委員-劉校生
中文關鍵字 蛋白質去磷酸化 
英文關鍵字 c-Jun/Sp1 interaction 
學科別分類
中文摘要 有文獻報導指出,轉錄因子的修飾對基因調控是重要的。在近年來我們實驗室的研究中,已發現當A431細胞處理EGF時,其能夠藉由活化Ras-ERK及Ras-Rac-JNK等路徑來誘導12(S)-lipoxygenase基因的表現,並且也可增加c-Jun的生合成。除此之外,我們發現12(S)-lipoxygenase promoter上並沒有AP1結合位置,但過度表現c-Jun時能夠誘導12(S)-lipoxygenase基因的表現,並且c-Jun/Sp1 interaction對12(S)-lipoxygenase基因的表現是很重要的。於是我們便有興趣來探討調控c-Jun/Sp1 interaction的機制為何。當細胞處理phosphatase抑制劑(cyclosporin A及cantharidin)時,發現這些抑制劑會抑制掉EGF誘導的12(S)-lipoxygenase mRNA表現及酵素活性;除此之外,這些抑制劑還能抑制EGF誘導的c-Jun/Sp1 interaction,但對c-Jun的生合成並沒有影響。由以上結果可知,c-Jun/Sp1 interaction可能會受到磷酸化與去磷酸化的調控。之前文獻曾報導過TAM-67,其為將c-Jun N端 truncated的突變型,可以和Sp1產生交互作用,我們便推測c-Jun C端的去磷酸化對調控c-Jun/Sp1 interaction可能扮演一個重要的角色。我們利用myc- TAM-67來探討是否EGF會誘導c-Jun C端進行去磷酸化。在免疫沉澱實驗中,我們發現EGF會誘導myc- TAM-67進行去磷酸化,且去磷酸化現象會隨著EGF處理時間增加而更明顯。綜合以上結果,我們可以知道,EGF會誘導c-Jun C端進行去磷酸化,以促使c-Jun和Sp1產生交互作用,進而促使12(S)-lipoxygenase基因的表現。
英文摘要 It is reported that modification of transcriptional factor is important for gene regulation. In recent years, we have found that EGF induces 12(S)-lipoxygenase gene expression via activation of Ras-Rac-JNK and Ras-ERK pathways in A431 cells, and also increases c-Jun biosynthesis. Furthermore, We found that 12(S)-lipoxygenase promoter without AP1 consensus binding site is induced by c-Jun overexpression, and the c-Jun/Sp1 interaction plays an important role in EGF-induced 12(S)-lipoxygenase gene expression. Therefore, we are interested in studying the regulation of c-Jun/Sp1 interaction. When A431 cells were treated with phosphatase inhibitors(cyclosporin A and cantharidin), the effect of EGF-induced expression of 12(S)-lipoxygenase mRNA and enzyme activity was inhibited. Furthermore, cyclosporin A and cantharidin also inhibited the interaction between c-Jun and Sp1 in EGF-treated cells while EGF-induced c-Jun biosynthesis was not affected. These results indicated that the interaction between c-Jun and Sp1 might be regulated by a phosphorylation/dephosphorylation mechanism. It was previously reported that TAM-67, which is c-Jun N-terminus truncated form, could interact with Sp1, and suggested that c-Jun C-terminus dephosphorylation may play an important role in the regulation of c-Jun/Sp1 interaction. We used myc-TAM-67 to study that whether EGF induces c-Jun C-terminus dephosphorylation. In immunopreciptation assay, we found that EGF induced dephosphorylation of myc-TAM-67 in a time-dependent manner. Taken together, EGF induces c-Jun C-terminus dephosphorylation and promotes c-Jun/Sp1 interaction, followed by the activation of 12(S)-lipoxygenase gene expression.
論文目次 目錄

中文摘要……………………………………………………………………1

英文摘要……………………………………………………………………2

縮寫檢索表…………………………………………………………………3

第一章 緒論
A. 前言…………………………………………………………...4
B. Lipoxygenase之分類………………………………………...5
C. Lipoxygenase之生理意義…………………………………...5
D. EGF之訊息傳遞路徑……………………………………......6
E. 磷酸化與去磷酸化對c-Jun及Sp1之影響……………….....7
F. Protein phosphatase及CKII之介紹………………………..8
G. 本論文之研究目標…………………………………………...9

第二章 實驗材料………………………………………………………10

第三章 實驗方法………………………………………………………16

第四章 實驗結果
A. 探討phosphatase是否參與在EGF誘導
12(S)-lipoxygenase基因表現的機制中…………………..38
B. 探討phosphatase如何調控12(S)-lipoxygenase
基因表現………………………………………….………...39
C. 探討c-Jun的C-端去磷酸化對於
12(S)-lipoxygenase基因表現的影響……………………..39

第五章 總結與討論……………………………………………………42

參考文獻………………………………………………………………….45

附圖……………………………………………………………………….53

附表……………………………………………………………………….68


參考文獻 參考文獻

Armstrong, S. A., Barry, D. A., Leggett, R. W. and Mueller, C. R. Casein kinase II-mediated phosphorylation of the C-terminus of Sp1 decreases its DNA binding activity. J. Biol. Chem. 272: 13489-13495, 1997.

Blaine, S., Wick, M., Dessev, C. and Nemenoff, R. A. Induction of cPLA2 in lung epithelial cells and non-small cell lung cancer is mediated by Sp1 and c-Jun. J. Biol. Chem. 276: 42737-42743, 2001.

Bollen M., and Beullens M. Signaling by protein phosphatases in the nucleus. Trends cell Biol. 12: 138-145, 2002.

Borgger, P., Hamberg, M., and Samuelsson, B. Transformation of arachidonic acid and homo-gamma-linolenic acid by rabbit polymorphonuclear leukocytes. Monohydroxy acids from novel lipoxygenase. J. Biol. Chem. 251: 7816-7820, 1976.

Boyle, W. J., Smeal, T., Defize, L. H., Angel, P., Woodgett, J. R., Karin, M., and Hunter, T. Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity. Cell 64: 573-584, 1991.

Capdevila, J., Yadagiri, P., Manna, S., and Faick, J. R. Absolute configuration of the hydroeicosatetraenoic acid (HETEs) formed during catalytic oxygenation of arachidonic acid by microsomal cytochrome p450. Biochem. Biophy. Res. Commun. 141: 10071-10110, 1986.

Chang, W. C. Cell signaling and gene regulation of human 12(S)-lipoxygenase expression. Prost. Other Lipid Med. 71: 277-285, 2003.

Chang, W. C., Liu, Y. W., Ning, C. C., Suzuki, H., Yoshimoto, T., and Yamamoto, S. Induction of arachidonate 12-lipoxygenase mRNA by epidermal growth factor in A431 cells. J. Biol. Chem. 268: 18734-18739, 1993.

Chang W. C., Nakao, J., Orimo, H., Tai, H. H., and Murota, S. I. Stimulation of 12-lipoxygenase activity in rat platelet by 17β-estradiol. Biochem. Pharmacol. 31: 2633-2638, 1982.

Clark, J. D., Schievella, Ar., Nalefaski, E. A., and Lin, L. L. Cytosolic phospholipase A2. J. Lipid Mediat. Cell signal. 12: 83-117, 1995.

Chang, W. C., Ning, C. C., Lin, M. T., and Huang, J. D. Epidermal growth factor enhances a microsomal 12-lipoxygenase activity in A431 cells. J. Biol. Chem. 267: 3657-3666, 1992.

Chen, B. K. and Chang, W. C. Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase. Proc. Natl. Acad. Sci. USA 97: 10406-10411, 2000.

Chen, L. C., Chen, B. K., Liu, Y. W., and Chang, W. C. Induction of 12-lipoxygenase expression by transforming growth factor-α in human epidermoid carcinoma A431 cells. FEBS Lett. 455: 105-110, 1999.

Cieslik, K., Lee, C. M., Tang, J. L., and Wu, K. K. Transcriptional regulation of endothelial nitric-oxide synthase by an interaction between casein kinase 2 and protein phosphatase 2A. J. Biol. Chem. 274: 34669-34675, 1999.

Cohen, P. T. Novel protein serine/threonine phosphatases: variety is the spice of life. Trends Biochem. Sci. 22: 245-251, 1997.

Engels, F., and Nijkamp, F. P. Pharmacological inhibition of leukotriene actions. Pharm. World Sci. 20: 60-65, 1998.

Furstenberger, G., Hagedom, H., Jacobi, T., Besemelder, E., Stephan, M., Lehmann, W. D., and Marks, F. Characterization of an 8-lipoxygenase activity induced by the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate in mouse skin in vivo. J. Biol. Chem. 266: 15738-15745, 1991.

Gao, X., Grignon, D. J., Chbihi, T., Zacharek, A., Chen, Y. Q., Sakr, W., Porter, A. T., Crissman, J. D., Pontes, J. E., and Powell, I. J. Elevated 12-lipoxygenase mRNA expression correlates with advanced stage and poor differentiation of human prostate cancer. Urology 46: 227-237, 1995.

Haidweger, E., Novy, M. and Rotheneder, H. Modulation of Sp1 activity by a cyclin A/CDK compex. J. Mol. Biol. 306: 201-212, 2001.

Han, K. K. and Martinage, A. Post-translational chemical modification(s) of proteins. Int. J. Biochem. 24: 19-28, 1992.

Hammarstrom, S., Hamberg, M., Samuelsson, B., Duell, E. A., Stawiski, M., and Voorhees, J. J. Increased concentrations of nonesterified arachidonic acid, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2alpha in epidermis of psoriasis. Proc. Natl. Acad. Sci. USA 72: 5130-5134, 1975.

Hussain, H., Shornick, L. P., Shannon, V. R., Wilson, J. D., Funk, C. D., Pentland, A. P., and Holtzman, M. J. Epidermis contains platelet-type 12-lipoxygenase that is overexpressed in germinal layer keratinocytes in psoriasis. Am. J. Physiol. 266: C243-C253, 1994.

Issinger, O.-G. Casein kinases: pleiotropic mediators of cellular regulation. Pharmacol. Ther. 59: 1–30, 1993.

Izumi, T., Hoshiko, S., Radmark, O., and Samuelsson, B. Cloning of the cDNA for human 12-lipoxygenase. Proc. Natl. Acad. Sci. USA 87: 7477-7481, 1990.

Kardassis, D., Papakosta, P., Pardali, K., and Moustakas, A. c-Jun transactivates the promoter of the human p21(WAF1/Cip1) gene by acting as a superactivator of the ubiquitous transcription factor Sp1. J. Biol. Chem. 274: 29572-29581, 1999.

Lin, A., Frost, J., Deng, T., Smeal, T., al-Alawi, N., Kikkawa, U., Hunter, T., Brenner, D., and Karin, M. Casein kinase II is a negative regulator of c-Jun DNA binding and AP-1 activity. Cell 70: 777-789, 1992.

Lin, L. L., A. Y., and Knopf, J. L. Cytosolic phospholipid A2 is coupled to hormonally regulated release of arachidonic acid. Proc. Natl. Acad. Sci. USA 89: 6149-6151, 1992.

Liu, Y. W., Asaoka, Y., Suzuki, H., Yoshimoto, T., Yamamoto, S., and Chang, W. C. Induction of 12-lipoxygenase expression by epidermal growth factor is mediated by protein kinase C in A431 cells. J. Pharmacol. Exp. Ther. 271: 567-573, 1994.

May, G. H., Allen, K. E., Clark, W., Funk, M., and Gillespie, D. A. Analysis of the interaction between c-Jun and c-Jun N-terminal kinase in vivo. J. Biol. Chem. 273: 33429-33435, 1998.

Melnikova, I. N. and Gardner, P. D. The signal transduction pathway underlying ion channel gene regulation by SP1-c-Jun interaction. J. Biol. Chem. 276: 19040-19045, 2001.

Mestres, P., Boldyref, B., Ebensperger, C., Hameister, H. & Issinger,O.-G. Expression of casein kinase 2 during mouse embryogenesis. Acta Anat. (Basel) 149: 13–20, 1994.

Miyamoto, T., Ogino, N., Yamamoto, S., and Hayaishi, O. Purification of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. J. Biol. Chem. 251: 2629-2636, 1976.

Münstermann, U., Fritz, G., Seitz, G., Yiping, L., Schneider, H. & Issinger, O.-G. Casein kinase II is elevated in solid tumours and rapidly non-proliferating tissue. Eur. J. Biochem. 189: 251–257, 1990.

Morton S., Davis, R. J., McLaren A., and Cohen P. A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun. EMBO J. 22: 3876-3886, 2003.

Narumiya, S., Salmon, J. A., Cottee, F. H., Weatherley, B. C., and Flower, R. T. Arachidonic acid 15-lipoxygenase from rabbit peritoneal polymorphonuclear leukocytes. Partial purification and properties. J. Biol. Chem. 256: 9583-9592, 1981.

Nikolakaki, E., Coffer, P. J., Hemelsoet, R., Woodgett, J. R. and Defize, L. H. K. Glycogen synthase kinase 3 phosphorylates Jun family members in vitro and negatively regulates their transactivating potential in intact cells. Oncogene 8: 833-840, 1993.

Papavassiliou, A. G., Treier, M. and Bohmann, D. Intramolecular signal transduction in c-Jun. EMBO J. 14: 2014-2019, 1995.

Piomelli, D., Volterra, A., Dale, N., Siegelbaum, S. A., Kandel, E. R., Schwartz, J. H., and Belardetti, F. Lipoxygenase metabolites of arachidonic acid as second messengers for presynaptic inhibition of Aplysia sensory cells. Nature 328: 38-43, 1987.

Pulverer, B. J., Kyriakis, J. M., Avruch, J., Nikolakaki, E., and Woodgett, J. R. Phosphorylation of c-jun mediated by MAP kinases. Nature 353: 670-674, 1991.

Sekiya, F., Takagi, J., Usui, T., Kawajiri, K., Kobayashi, Y., Sata, F., and Saito, Y. 12(S)-Hydroxyeicosatetraenoic acid play a central role in the regulation of platelet activation. Biochem. Biophys Res. Commun. 179: 345-351, 1991.

Shibasaki, F., Price, E. R., Milan, D., and McKeon, F. Role of kinases and the phosphatase calcineurin in the nuclear shuttling of transcription factor NF-AT4. Nature 382: 370-373, 1996.

Tang, D. G., Chen, Y. Q., and Honn, K. V. Arachidonate lipoxygenases as essential regulators of cell survival and apoptosis. Proc. Natl. Acad. Sci. USA 93: 5241-5246, 1996.

Tang, D. G., and Honn, K. V. 12-Lipoxygenase, 12(S)-HETE, and cancer metastasis. Ann. N. Y. Acad. Sci. 744: 199-215, 1994.

Tarnawski, A., Stachura, J., Durbin, T., Sarfeh, I. J., and Gergely, H. Increased expression of epidermal growth factor receptor during gastric ulcer healing in rats. Gastroenterology 102: 695-698, 1992.

Walker, F., Kato, A., Gonez, L. J., Hibbs, M. L., Pouliot, N., Levitzki, A., and Burgess, A. W. Activation of the Ras/mitogen-activated protein kinase pathway by kinase-defective epidermal growth factor receptors results in cell survival but not proliferation. Mol. Cell. Biol. 18: 7192-7204, 1998.

Winitiz, S., Gupta, S. K., Cian, N., Heasley, L. E., Nemenoff, R. A., and Johnson, G. L. Expression of Gi2α subunit inhibits ATP and thrombin stimulation of cytoplasmic phospholipase A2-mediated arachidonic acid release independent of Ca++ and mitogen-activated protein. J. Biol. Chem. 269: 1889-1895, 1994.

Wright, N. A., Pike, C., and Elia, G. Induction of a novel epidermal growth factor-secreting cell lineage by mucosal ulceration in human gastrointestinal stem cells. Nature 343: 82-85, 1990.

Van Leyen, K., Duvoisin, R. M., Engelhardt, H., and Wiedmann, M. A function for lipoxygenase in programmed organelle degradation. Nature 395: 392-395, 1998.

Xu, X. X., Rock, C. O., Qin, Z., Leslie, C. C., and Jackowski, S. Regulation of cytosolic phospholipase A2 phosphorylation and eicosanoid production by colony-stimulating factor. J. Biol. Chem. 269: 31693-31700, 1994.

Yamamoto, S. Mammalian lipoxygenase: molecular structures and functions. Biochim. Biophys. Acta. 1128: 117-131, 1992.

Zhang, S., and Kim, K. H. Protein kinase CK2 down-regulates glucose-activated expression of the acetyl-CoA carboxylase gene. Arch. Biochem. Biophys. 338: 227-232, 1997.
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2003-12-29起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2003-12-29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