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系統識別號 U0026-0812200913540479
論文名稱(中文) 探討NDPK-A 與eEF1Bα的交互作用
論文名稱(英文) An interaction of NDP kinase A/NM23-H1 with eEF1Bα elongation factor
校院名稱 成功大學
系所名稱(中) 分子醫學研究所
系所名稱(英) Institute of Molecular Medicine
學年度 95
學期 2
出版年 96
研究生(中文) 朱小瓀
研究生(英文) Hsiao-Ju chu
電子信箱 pig191919@seed.net.tw
學號 T1694401
學位類別 碩士
語文別 英文
論文頁數 55頁
口試委員 指導教授-張玲
指導教授-蘇五洲
口試委員-吳梨華
口試委員-蔣輯武
中文關鍵字 腫瘤轉移  轉譯延長  核苷二磷酸激酶A 
英文關鍵字 Translation elongation  Nucleoside diphosphate kinase A  Metastasis  NM23-H1 
學科別分類
中文摘要 腫瘤轉移是一個複雜且多步驟連續性的主動的過程,腫瘤細胞從原發
腫瘤向遠端器官擴散及其隨後的生長而導致腫瘤的擴散。NM23-H1 最初
是在乳癌等腫瘤中發現是一個腫瘤轉移抑制基因(metastasis suppressor
gene),其所轉錄轉譯成的蛋白質是核苷二磷酸激酶A(nucleoside
diphosphate kinase A, NDPK-A)。然而,在神經母細胞瘤和其他侵犯性腫
瘤細胞中發現NDPK-A 表現會促進腫瘤轉移。為了要了解NDPK-A 在腫
瘤轉移種的角色,我們實驗室先前利用酵母菌雙雜合系統(yeast
two-hybrid system)以NDPK-A 篩選到其結合蛋白-轉譯延長因子
(eEF1Bα)。在蛋白質合成轉譯作用中,eEF1Bα催化eEF1A 由GDP 結
合形式轉變成GTP 結合形式。在此, 我利用共同免疫沉澱法
( co-immunoprecipitation assay ) 證實: NDPK-A 可以在人類細胞
(HEK293T)中直接和eEF1Bα結合,且跟轉移有關的突變-NDPK-AS120G
和eEF1Bα結合力較強。在人工穩定表現NDPK-A 或NDPK-AS120G 的神
經母細胞中,皆發現eEF1Bα的表現量降低。這些發現也提供一個新的研
究方向,藉由NDPK-A 和eEF1Bα的結合,連結腫瘤轉移和轉譯作用。
英文摘要 Tumor metastasis is a complex process which involves the spread of
cancer cells from the origin to distant sites, where they cause secondary
lesions. Nucleoside diphosphate kinase A (NDPK-A) encoded by NM23-H1 is
originally proposed as a metastasis suppressor gene in certain cancer types,
including breast carcinoma. In contrast, NDPK-A behaves as a metastasis
promoter in other cancer types, such as neuroblastoma. To understand the
molecular mechanism by which NDPK-A contributes to metastasis, our
laboratory previously identified that a translation elongation factor, eEF1Bα,
interacts with NDPK-A in the yeast two-hybrid system. The eEF1Bα belongs
to the guanine nucleotide exchange protein family and catalyzes the exchange
of GDP- or GTP-bound eEF1A in the elongation cycle of protein biosynthesis.
In this study, I show a direct interaction of NDPK-A with eEF1Bα in human
embryonic kidney (HEK) 293T cells by co-immunoprecipitation. Interestingly,
the binding of eEF1Bα with metastasis-associated S120G mutation
(NDPK-AS120G) was stronger than its wild type. Moreover, overexpression of
NDPK-A or NDPK-AS120G reduces eEF1Bα expression in human
neuroblastoma NB69 cells. These finding provide a potential link between
metastasis and translation via the interaction of NDPK-A and eEF1Bα.
論文目次 I. INTRODUCTION.......................................................................................................6
I.i. Neuroblastoma................................................................................................... 6
I.ii. NM23/NDPK...................................................................................................... 7
I.iii. NDPK-A.............................................................................................................. 8
I.iv. Metastasis and NDPK-A................................................................................... 8
I.v. Translation ......................................................................................................... 9
I.vi. Translation elongation factor and tumorigenesis ......................................... 10
I.vii. Hypothesis ........................................................................................................ 11
II. METERIALS AND METHODS ..............................................................................12
II.i. Reagents............................................................................................................ 12
II.ii. Construction of expression of expression plasmids ...................................... 13
II.iii. Cell culture ....................................................................................................... 15
II.iv. Transfection...................................................................................................... 16
II.v. Western blot analysis....................................................................................... 16
II.vi. Co-immunoprecipitation................................................................................. 18
II.vii. Generation of GTP- and GDP-bound eEF1A protein.................................. 19
III. RESULTS ................................................................................................................... 20
III.i. Construction and expression of NDPK-A and NDPK-AS120G ...................... 20
III.ii. Construction of wild type and deletion mutants of eEF1Bα ....................... 20
III.iii. Construction of the pGEX-2TK-NDPK-AH118F expression plasmids.......... 21
III.iv. Ectopic expression of NDPK-A or eEF1Bα in HEK293T cell ..................... 21
III.v. Co-expression of NDPK-A and eEF1Bα in HEK293T cell .......................... 22
III.vi. NDPK-A interacts with eEF1Bα elongation factor in HEK293T cells ....... 22
III.vii. Effects of GTP and GDP on the interaction of eEF1A, eEF1Bα and
NDPK-A............................................................................................................ 23
III.viii. Effect of NDPK-A alterations the expression of endogenous eEF1Bα in
stable transfectants derived from human neuroblastoma NB69 cell.......... 23
IV. DISSCUSSION .......................................................................................................... 25
V. REFERENCE ............................................................................................................28
VI. TABLES...................................................................................................................... 36
Table 1. The international neuroblastoma staging system (INSS) [2] ............... 36
Table 2. The phosphotransferase activity of NDPK............................................ 37
5
Table 3. Band intensity ratios of eEF1A, eEF1Bα and NDPK-A in the
immunoprecipitation complexes. .......................................................... 38
VII. FIGURES ................................................................................................................... 39
Figure 1. Construction of the pCMV-Tag1-NDPK-A expression plasmid......... 39
Figure 2. Schematic structures of the wild type and deletion mutants of
eEF1Bα .................................................................................................... 40
Figure 3. Construction of the pCMV-Tag1-eEF1A, pCMV-Tag1-N-eEF1Bα,
pCMV-Tag1-C-eEF1Bα expression plasmids. ..................................... 41
Figure 4. Restriction enzyme digestion patterns of pCMV-Tag1-NDPK-A,
pCMV-Tag1-eEF1Bα and the deletion mutants. ................................. 42
Figure 5. Direct DNA sequencing of the pCMV-Tag1-NDPK-A,
pCMV-Tag1-eEF1Bα, pCMV-Tag1-N-eEF1Bα,
pCMV-Tag1-C-eEF1Bα constructs....................................................... 44
Figure 6. Restriction enzyme patterns of the pCMV-Tag1-NDPK-AS120G and
pCMV-Tag1-NDPK-AH118F plasmids. ................................................... 45
Figure 7. Schematic presentation of pGEX-2TK-eEF1Βα, pGEX-2TK-H118F,
pGEX-2TK-eEF1A1 constructs. ........................................................... 46
Figure 8. Confirmation of the pGEX-2TK-NDPK-AH118F construct. ................. 47
Figure 9. Direct DNA sequencing of the pGEX-2TK-NDPK-AH118F constructs.
.................................................................................................................. 48
Figure 10. Western blot analysis of ectopic and endogenous NDPK-A,
eEF1Bα, N-eEF1Bα and C-eEF1Bα expressed in HEK293T cell...... 49
Figure 11. Western blot analysis of co-expressed NDPK-A, NDPK-AS120G and
eEF1Bα in HEK293T cells..................................................................... 50
Figure 12. Interaction of NDPK-A with eEF1Bα in HEK293T cell. .................... 51
Figure 13. Immunoprecipitation of NDPK-A, NDPK-AS120G or NDPK-AH118F
with eEF1Bα in the presence of GTP or GDP...................................... 54
Figure 14. Effects of NDPK-A variants on the expression of eEF1A and
eEF1Bα in NB69- and HEK293T-derivatives. ..................................... 55
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