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研究生(外文):Tsai-Hwa Jong
論文名稱(外文):Identification of N-terminal Eps8 binding proteins by Yeast Two-Hybrid Method
指導教授(外文):Tzeng-Horng Leu
外文關鍵詞:Eps8Yeast Two-Hybridprotein
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之前我們實驗室已經證實在老鼠纖維母細胞中過量表達p97Eps8會造成細胞的轉型及促進腫瘤的產生,所以可以知道p97Eps8在細胞生理功能上扮演著重要的角色。由胺基酸序列的分析得知p97Eps8具有:(1) N端的split PH domain □ (2) 一個SH3 domain □ (3) 四個proline rich regions □ 及 (4) 一個putative nuclear targeting sequence。為了進一步了解p97Eps8和腫瘤之間的關係,我們希望能有系統的分析出p97Eps8的結合蛋白質。在此我們利用yeast two-hybrid的方法尋找可以和N-terminal p97Eps8 結合的蛋白質。從human brain cDNA library我們篩選出246個可以製出和N-terminal p97Eps8相互結合之蛋白質的cDNA。其中的NEBP1(N-terminal Eps8 Binding Protein 1) 由GenBank的基因庫比對為BAIAP2。NEBP2為TLE2。
由於先前實驗室的研究結果指出p97Eps8會被non-receptor tyrosine kinase Src所磷酸化,同時97Eps8的proline rich regions在in vitro可以和Src的SH3 domain結合。所以我們也利用yeast two-hybrid的方法定出p97Eps8真正可以和Src的SH3 domain結合的胺基酸序列。實驗結果我們發現完整的wt-p97Eps8及261-Eps8可以和Src的SH3SH2 domain結合,而SrcSH3SH2 domain無法與其他truncated p97Eps8結合。
Previously, we demonstrated that cells overexpressing p97Eps8 not only exhibited the ability of focus formation in cell culture but also promoted the tumor formation in mice as compared to controls. However the N-terminal truncated 261-p97Eps8 has lost such transforming ability. Thus, the N-terminal domain may play an important role in Eps8-mediated cellular transformation. Protein sequence analysis revealed the following domains existing in the N-terminal p97Eps8 that might contribute to its cellular functions : a putative nuclear targeting sequence, a split PH domain and two proline-rich regions. In this study we utilized yeast two-hybrid method to screen a human brain cDNA expression library to search for N-terminal p97Eps8 binding proteins (NEBP). We have obtained 246 cDNA clones encoding the potential N-terminal p97Eps8 binding proteins. Sequence comparison with the information derived from GenBank, NEBP1 and NEBP2 were identified BAIAP2 and TLE2 respectively. How these proteins interact with p97Eps8 and play a role in Eps8-induced cellular transformation needs further investigation.
Previously, there was evidence indicating that Src can phosphorylate p97Eps8 and the proline rich regions of p97Eps8 can interact with the Src SH3 doamin in an in vitro binding assay. To substantiate this interaction in vivo, we also utilize yeast two-hybrid method to define the Src SH3 domain binding sequence of p97Eps8. We find that SrcSH3SH2 domain can interact with wt-p97Eps8 and 261-p97Eps8, but not the other truncated p97Eps8.
第一章 緒論7
第二章 實驗材料及方法15
第一節 實驗材料16
第二節 實驗方法21
第三章 實驗結果32
第四章 討論38
參考文獻 (References)
Alvarez C. E., Sutcliffe J. G., and Thomas E. A. Novel isoform of insulin receptor substrate p53/p58 is generated by alternative splicing in the CRIB/SH3-binding region. J. Biol. Chem. 277: 24728-24734, (2002).
Di Fiore P. P., and Scita G. Eps8 in the midst of GTPases. The Intermational J. Biochem. Cell Biol. 34: 1178-1183, (2002).
Fazioli F., Minichiello L., Matoska V., Castagnino P., Miki T., Wong W. T., and Di Fiore P. P. Eps8, a substrate for the epidermal growth factor receptor kinase, enhances EGF-dependent mitogenic signals. EMBO J. 12: 3799-3808, (1993).
Gallo R., Provenzano C., Carbone R., Di Fiore P. P., Castellani L., Falcone G., and Alema S. Regulation of the tyrosine kinase substrate Eps8 expression by growth factors, v-Src and terminal differentiation. Oncogene 15: 1929-1936, (1997).
Govind S., Kozma R., Monfries C., Lim L., and Ahmed S. Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing the 58-kd insulin receptor substrate to filamentous actin. J. Cell Biol. 152: 579-594, (2001).
Grbavec D., Lo R., Liu Y., and Stifani S. Transducin-like Enhancer of split 2, a mammalian homologue of drosophila groucho, acts as a transcriptional repressor, interacts with Hairy/Enhancer of split proteins, and is expressed during neuronal development. Eur. J. Biochem. 258: 339-349, (1998).
Iba H., Cross F. R., Garber E. A., and Hanafusa H. Low level of cellular protein phosphorylation by nontransforming overproduced p60c-Src. Mol. Cell Biol. 5: 1058-1066, (1985).
Irby R. B., and Yeatman T. J. Role of Src expression and activation in human cancer. Oncogene 19: 5636-5642, (2000).
Jacobs C., and Rubsamen H. Expression of pp60c-Src protein kinase in adult and fetal human tissue:high activities in some sarcomas and mammary carcinomas. Cancer Res.. 43: 1696-1702, (1983).
Karlsson T., Songyang Z., Landgren E., Lavergene C., and Di Fiore P. P. Molecular interactions of the Src homology 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain proteins. Oncogene 10: 1475-1483, (1995).
Kishan K. V. R., Scita G., Wong W. T., Fiore P. P. D., and Newcomer M. E. The SH3 domain of eps8 exists as a novel interwined dimmer. Nat. Struct. Biol. 4: 739-743, (1997).
Krugmann S., Jordens I., Gevaert K., Driessens M., Vandekerckhove J., and Hall A. Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex. Current Biol. 11: 1645-1655, (2001).
Lemmon M. A., Ferguson K. M., and Abrams C. S. Pleckstrin homology domains and the cytoskeleton. FEBT Letters 513: 71-76, (2002).
Maa M. C., Lai J. R., Lin R. W., and Leu T. H. Enhancement of tyrosyl phosphorylation and protein expression of eps8 by v-Src. Biochim Biophys Acta. 1450: 341-351, (1999).
Maffucci T., and Falasca M. Specificity in pleckstrin homology (PH) domain membrane targeting: a role for a phosphoinositide-protein co-operative mechanism. FEBS Letters 506: 173-179, (2001).
Matoskova B., Wong W. T., Nomura N., Robbins K. C., and Di Fiore P. P. RN-tre specifically binds to the SH3 domain of eps8 with high affinity and confers growth advantage to NIH3T3 upon carboxy-terminal truncation. Oncogene 12: 2679-2688, (1996).
Matoskova B., Wong W. T., Salcini A. E., Pelicci P. G., and Di Fiore P. P. Constitutive phosphorylation of eps8 in tumor cell lines: relevance to malignant transformation. Mol. Cell. Biol. 15: 3805-3812, (1995).
Miki H., and Takenawa T. WAVE2 serves a functional partner of IRSp53 by regulating its interaction with Rac. BBRC 293: 93-99, (2002).
Provenzano C., Gallo R., Carbone R., Di Fiore P. P., Falcone G., Castellani L., and Alema S. Eps8, a tyrosine kinase substrate, is recruited to the cell cortex and dynamic F-actin upon cytoskeleton remodeling. Exp.Cell Res. 242: 186-200, (1998).
Rosalyn B Irby and Timothy J Yeatman. Role of Src expression and activation in human cancer. Oncogene 19: 5636-5642, (2000).
Scita G., Nordstrom J., Carbone R., Tenca P., Giardina G., Gutkind S., Bjarnegard M., Betsholtz C., and Di Fiore P. P. Eps8 and E3b1 transduce signals from Ras to Rac. Nature 401: 290-293, (1999).
Stifani S., Blaumeller C. M., Redhead N. J., Hill R. E., and Artavanis-Tsakonas S. Human homologs of a Drosophila Enhancer of split gene product define a novel family of nuclear proteins. Nat. Genet. 2:119-127, (1992).
Tuko Okamura-Oho, Miyashita T., and Yamada M. Distinctive tissue distribution and Phosphorylation. Biochemical and Biophysical Research Communications 289:957-920, (2001).
Verbeek B. S., Vroom T. M., Adriaansen-Slot S. S., Ottenhoff-Kalff A. E., Geertzema J. G. N., Hennipman A., and Rijksen G. c-Src protein expression is increased in human breast cancer. An immunohistochemical and biochemical analysis. J. Pathology 180: 383-388, (1996).
Wiener J. R., Nakano K., Kruzelock R. P., Bucana C. D., Bast R. C., and Gallick G. E. Decreased Src tyrosine kinase activity inhibits malignant human ovarian cancer tumor growth in a nude mouse model. Clinical Cancer Research 5: 2164-2170, (1999).
Yeh T. C., Ogawa W., Danielsen A. G., and Roth R. A. Characterization and cloning of a 58/53-kDa substrate of the insulin receptor tyrosine kinase. J. Biol. Chem. 271: 2921-2928, (1996).
Yuko Okamura-Oho, Milyashita T., Ohmi K., and Yamada M. Dentatorubral-pallidoluysian atrophy protein interacts through a proline-rich region near polyglutamine with the SH3 domain of an insulin receptor tyrosine kinase substrate. Human Molecular Genetics 8: 947-957, (1999).
林妲妮 建立持續性表達附著於細胞膜且不具完整Pleckstrin Homology p97Eps8的細胞株及其對細胞轉型的影響。 國立成功大學藥理學研究所碩士論文。中華民國九十年七月。
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