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系統識別號 U0026-0308201518530300
論文名稱(中文) 研究v-HRas 泛素化後的作用
論文名稱(英文) Functional study of v-HRas ubiquitination
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 103
學期 2
出版年 104
研究生(中文) 高國允
研究生(英文) Guo-Yun Kao
學號 S26024094
學位類別 碩士
語文別 英文
論文頁數 47頁
口試委員 指導教授-呂增宏
口試委員-馬明琪
口試委員-洪建中
中文關鍵字 v-H-Ras  Eps8  泛素化  細胞生長 
英文關鍵字 v-H-RAS  Eps8  ubiquitination  cell growth 
學科別分類
中文摘要 Ras家族蛋白屬於小型GTP 結合蛋白的其中一員,其序列上帶有effector domain可以去調控訊息傳遞以及C端上的hypervariable region則可以去調控他們在細胞膜上的位置。他們也會去調控許多細胞生長的功能,像是增生能力、分化能力以及存活能力。而Ras的泛素化則會去影響其蛋白質本身的穩定程度以及蛋白質在細胞裡的位置。在近期的研究已經顯示說在Ras117位點和147位點進行單一泛素化會去增加Ras蛋白的活性。然而在Ras蛋白上的其他離胺酸位點的功能尚未被釐清。因此,在我們的研究中,我們想要在vH-Ras上找尋有潛力的泛素化位點以及研究他們在細胞生長上所扮演的角色。我們首先將C端hypervariable region 上的第167、170以及185離胺酸位點突變成精氨酸 (簡稱C3KR)。此外,因為第42離胺酸位點位於switch I and II domain,所以我們也將N端上的第5、42以及147離胺酸位點突變成精氨酸 (簡稱N3KR)。在我們結果顯示C3KR並不會抑制蛋白的泛素化,而是C3KR的多泛素化程度高於Wt進而造成vH-Ras的降解。我們也發現在給予cycloheximide (CHX)之後,其C3KR和N3KR的v-H-Ras降解程度高於Wt。有趣的是,在H-Ras knockdown T-24細胞中發現其細胞中的Eps8表現量和ERK活性會下降。為了釐清H-Ras如何影響Eps8和ERK 活性,我們將C3KR放入HeLa細胞中;結果顯示C3KR可以促進Eps8的降解以及抑制ERK活性。此外,我們發現在HeLa細胞和穩定H-Ras knockdown T-24細胞中表現C3KR也會降低細胞在軟瓊脂生長的速度。由以上結果我們可以得知C端上的離胺酸殘基會去調節H-Ras和Eps8的蛋白穩定性進而去影響細胞的生長速度。
英文摘要 Ras family proteins, a class of small GTP-binding proteins, contain effector domain for its signal transduction and the C-terminal hypervariable region that regulate their plasma membrane localization. Ras regulate many biological functions, including proliferation, differentiation, and survival. Ubiquitination of Ras has been implicated in its protein stability and cellular localization. Recent studies further indicate that mono─ubiquitination increases Ras protein activity by Ras 117 and 147 lysine (K). However, the function of other lysine residues on Ras protein is still unclear. In this study, we searched for potential ubiquitination residues on vH-Ras and studied their biological role. We first generated K-R (arginine) mutation in the C-terminal hypervariable region on H-Ras, i.e. 167K, 170K and 185K (designated as C3KR). Because 42K is located between Switch I and II domain, we also generated mutation in the N-terminal region, i.e.5K, 42K and 147K (designated as N3KR). Unexpectedly, instead of reducing protein ubiquitination, our data indicated that C3KR mutation exhibited more poly-ubiquitinated H-Ras than in wild types, and resulted in v-HRAS degradation. Also, we found that v-HRas degradation in C3KR and N3KR mutant types was higher than in wild types with cycloheximide (CHX) treatment. Interestingly, EPS8 and ERK activity was downregulation in H-Ras knockdown T-24 cells. To clarify how H-Ras affects Eps8 and ERK activity, we generated C3KR in HeLa cells to confirm this question. The data show that C3KR can promote Eps8 degradation and reduce ERK activity. Moreover, we found that C3KR mutant form also reduced cell colony numbers in HeLa cells and stable H-Ras knockdown T-24 cells growing in soft agar. Taken together, our results point out C-terminal lysine residues regulated the protein stability of H-Ras and Eps8 and in turn affected colony formation.
論文目次 Abstract in Chinese....... i

Abstract......... iii

Acknowledgement........ . v

Table of Contents........ . vi

List of figures........vii

Abbreviations......... viii

Introduction........1

Materials and Method........11

Results........ 20

Discussion........24

References........28

Appendix...........47
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