||探討癌細胞中WWP2 與 Eps8 之間的交互作用
||Functional study of the interaction between WWP2 and Eps8 in cancer cell
||Department of Pharmacology
WWP2 (WW domain containing E3 ubiquitin protein ligase 2)，又被稱為 atrophin-1-interacting protein 2 (AIP-2)，具有 E3 ligases 活性，屬於 NEDD4-like protein 家族中的其中一員。過去的報導指出 WWP2 會調控癌細胞的細胞增生，但是調控的機制並不完全瞭解。Eps8 (Epidermal growth factor receptor pathway substrate No.8) 為表皮細胞生長因子受體 (EGFR) 與非受體酪氨酸激酶 (Src) 的共同受體。 Eps8 調控細胞的生長與移動性，在癌細胞中過度的表達 Eps8 會促進細胞的增生。我們實驗室先前的研究發現 Eps8 可能為 WWP2 的結合蛋白， Eps8 會與 WWP2 有交互作用，並且會伴隨多泛素化的現象產生。然而，Eps8 如何與 WWP2 結合，以及這個結合會造成什麼結果還是未知的。因此，我們想了解 Eps8 與 WWP2 之間交互作用的情形，以及 WWP2 在 Eps8 致癌過程中扮演什麼角色。從我們試管外 GST pull down 與免疫共沉澱法的實驗中發現 Eps8 的 PPY motif 能與 WWP2 的 WW domain 進行交互作用。進一步，我們發現在子宮頸癌HeLa 細胞中，當我們把Eps8 PPY 序列突變成 PAF 時會減弱 Eps8所調控的colon 形成 。所以 WWP2 與 Eps8 之間的交互作用在癌細胞的細胞生長中可能扮演一個重要的角色。
WWP2 (WW domain containing E3 ubiquitin protein ligase 2), also known as atrophin-1-interacting protein 2 (AIP-2), is a member of the NEDD4-like E3 ligases family. It has been indicated to regulate cell proliferation in cancer cells, but the underlying mechanism is still unknown. Eps8 (Epidermal growth factor receptor pathway substrate No.8) is a substrate for both receptor Epidermal growth factor receptor (EGFR) and non-receptor tyrosine kinase (Src). Eps8 regulates cell motility and promotes cell proliferation in cancer cells. Our unpublished data indicates Eps8 may be a WWP2-binding protein and the associated Eps8 is polyubiquitinated. However, the biological function of WWP2-Eps8 interaction is unknown. In this study, we want to address the interacting domains between WWP2 and Eps8. Then, we would like to know whether WWP2 plays a role in Eps8-mediaed oncogenesis. Our data indicated that PPY motif in Eps8 was required to interact with the WW domain(s) in WWP2 by both in vitro GST pull down and co-immunoprecipitation experiments. Furthermore PAF mutation on the PPY sequence of Eps8 impaired Eps8-mediated colony formation in HeLa cell. Thus, WWP2-Eps8 interaction might play an important role in cell growth of cancer cells.
第一章 緒論 1
第二章 實驗材料及方法 6
第三章 實驗結果 22
第四章 討論 26
第五章 圖表 29
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