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系統識別號 U0026-0208201213270000
論文名稱(中文) 探討在v-Src轉型細胞中Eps8磷酸化對IRSp53交互作用與STAT3活化的角色
論文名稱(英文) Studying the role of tyrosyl phosphorylation of Eps8 in IRSp53 interaction and STAT3 activation in v-Src transformed 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 100
學期 2
出版年 101
研究生(中文) 鄭羽伸
研究生(英文) Yu-Shen Cheng
學號 s26991097
學位類別 碩士
語文別 英文
論文頁數 62頁
口試委員 指導教授-呂增宏
口試委員-馬明琪
召集委員-賴明德
口試委員-劉校生
中文關鍵字 Eps8  IRSp53  Stat3  Src  細胞增殖  磷酸化  轉型 
英文關鍵字 Eps8  IRSp53  Stat3  Src  Proliferation  phosphorylation  transformation 
學科別分類
中文摘要 Eps8(EGFR pathway substrate no.8)為receptor tyrosine kinase( EGFR)及non-receptor tyrosine kinas(v-Src)的受質。在許多的腫瘤細胞中,Eps8有持續磷酸化的現象。我們實驗室指出在v-Src調控之細胞轉型(IV5)中,抑制Eps8表達會減緩癌細胞的增生及腫瘤的形成。又發現Eps8-IRSp53交互作用參與在v-Src所誘導的細胞轉型,以及影響下游Stat3 Try705的磷酸化。因此我們想要探討在v-Src所調控的細胞轉型當中,Eps8的磷酸化是否會影響和IRSp53的交互作用及促使下游的Stat3活化。先前實驗室對Eps8 45、524、及674位點酪氨酸各別做點突變成苯丙胺酸,證明45及524位點是主要磷酸化的位點。因此分別將wild-type-Eps8、Phe-45-Eps8、Phe-524-Eps8、Phe-45/-524-Eps8分別送入Eps8弱化的IV5細胞中,以建立穩定表達的細胞株。我們研就證實Src各自磷酸化是在Tyr-45或是Tyr-524位置上。在細胞生長及soft agar assay實驗當中,我們發現Phe-45、Phe-524、Phe-45/-524-Eps8會抑制細胞增殖、colony formation、Stat3的Tyr-705位點的磷酸化。我們也證實在IV5細胞當中,Eps8確實有著高度的磷酸化及大量IRSp53的交互作用情形。進一步分析在個別磷酸化位點當中,Phe-45-Eps8、Phe-524-Eps8及Phe-45/-524-Eps8都會降低和IRSp53交互作用。總合上述,我們發現Src調控Eps8於Tyr-45及Tyr-524的磷酸化,藉由影響和IRSp53的交互作用,使下游Stat3活化,造成細胞不正常的分裂。
英文摘要 Eps8 (EGFR pathway substrate no.8) is a substrate of both receptor tyrosine kinase ( EGFR) and non-receptor tyrosine kinas(v-Src). Previously, we demonstrated that Eps8 knockdown decreased cell proliferation and tumorigenesis in v-Src transformed cell (IV5) and Src promoted the interaction between Eps8 and IRSp53, which contributed to Stat3 activation and cell transformation. We wondered whether Src-mediated phosphorylation on Eps8 facilitated the Eps8-IRSp53 interaction and the following Stat3 activation. To address this, we confirmed the tyrosine residues 45 and 524 on Eps8 as the major phosphorylated sites by Src. Then, we generated Eps8-knockdown IV5 cell expressing wild type-, F-45-, F-524-, or F-45/524-Eps8. We observed that Src-mediated Eps8 phosphorylation on Tyr-45 and Tyr-524 was in an independent manner. When we compared the cell proliferation and colony formation in soft agar among these cells, we observed mutation of either one of these tyrosine residues to phenylalanine on Eps8 abolished Src-mediated mitogenesis. In addition, the Eps8-IRSp53 interaction and Stat3 activation were also impaired in these mutated Eps8-expressing IV5 cells. Taken together, our data indicated that Src-mediated Eps8 phosphorylation enhanced the Eps8-IRSp53 interaction, resulting in the following Stat3 activation and increased cell proliferation in v-Src transformed cells.
論文目次 Abstract 1
中文摘要(Abstract in Chinese) 3
Acknowledgement 5
Content 7
Figure contents 9
Abbreviations 11
Introduction 13
Materials and Methods 22
Results 26
Discussion 36
References 39
Table 47
Figures 48
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