臺灣博碩士論文加值系統

中文摘要
磷酸酶Protein phosphatase 2A， 以下簡稱PP2A，屬於一種serine / threonine的磷酸酶。參與很多細胞內的調節作用，例如:參與細胞內的訊息傳遞、調節細胞週期的進行、及調控細胞的生長及細胞凋亡等等。而PP2A主要是由一個36-kDa的催化次單元，稱之為C次單元；及一個65-kDa的結構次單元，稱之為A次單元；以及具有調節功能的B次單元所組成的三元複合體。目前相信B次單元能夠決定PP2A的受質特異性 ，此外B次單元又區分為B(B55/PR55)、B’ (B56/PR61)、B” (PR72/PR130) 以及 B”’(striatin/ SG2NA) 四個不同的家族。而在B’家族中又包含 a、b、g、d 以及e 五種不同的異構型。B56g3則屬於B’ g 家族中的一個spliced variants。此外最近的研究證據指出：在一些人類的肺癌細胞中，會失去PP2A/ B56g的表現；同時又發現將PP2A/ B56g3送入細胞過度表現時，能夠減低這些癌細胞的癌化特性。因此這些證據暗示著： B56g3 次單元也許扮演一個調節細胞轉型的角色，而這個次單元的表現的變異也許會影響腫瘤的形成。因此我的研究方向，主要想探討B56g3 次單元在細胞中所扮演的調節功能為何，是否與PP2A扮演腫瘤抑制的角色有關呢？在NIH 3T3細胞及COS-7細胞中，利用轉染法結合免疫螢光染色的技術及GFP-tagging的方法去評估B56g3 次單元在細胞中的表現位置。我們發現B56g3 次單元主要集中在細胞核表現 ，更有趣的是：一些過度表現B56g3 次單元的細胞會有不正常的細胞型態，類似凋亡的細胞，並且有細胞核皺縮的情形。但是更有趣的是，我們發現在篩選的穩定表現B56g3 次單元的NIH 3T3細胞群，除了有些仍保持集中在核的表現外，大部分是呈現細胞核及細胞質均質表現，有少數甚至是在細胞質中有較多的表現。此外也發現過度表現B56g3 次單元的細胞，相較於pMSCV vector alone表現的細胞，會延遲NIH 3T3細胞的細胞生長。在進一步分析細胞週期分布的情形 ，發現到過度表現B56g3 次單元的細胞其G1、G2/M phase 會減少而S phase會增加，因此我們初步推測B56g3 次單元的大量表現可能會導致細胞週期的S-phase停滯。另一方面，當給予細胞serum withdrawl的刺激時 ，也發現過度表現的B56g3 次單元會增加細胞死亡現象。此外對於訊息傳遞的影響，則觀察到B56g3 次單元能夠明顯的降低激酶AKT在Threonine 308位點的磷酸化；但是對於Serine 473位點則沒有影響；另外對於extracellular signal –regulated kinases (Erk1/2) 並沒有顯著的影響。所以綜合上述的研究結果 ，我們發現B56g3 次單元的表現位置是可具變動性，而且可能與其調節的功能有關，當其過度表現時，延遲了細胞的生長，其可能的機制是透過調控S-phase停滯或是促成細胞凋亡所造成。

Abstract
Protein phosphatase 2A (PP2A) belongs to a family of serine/threonine phosphates, and is involved in a regulation of broad range of cellular processes, such as signal transduction, cell cycle regulation, cell growth and cell death. The holoenzyme of PP2A consists of a 36-kDa catalytic C subunit, a 65-kDa structural A subunit, and a variable regulatory B subunit, which is thought to determine the substrate specificity and subcellular localization of PP2A. At present, four different families of B subunit have been identified, termed B (B55/PR55), B’(B56/PR61) , B” (PR72/PR130, PR59, PR48), and B”’(striatin, SG2NA). Among these, the B’family contains at least five distinct gene products, denoted a, b, g, d and e, and B56g3 belongs to one of the spliced variants of B’g family. Recent evidence has shown that loss of expression of PP2A B56g in some human lung cancer cell lines, whereas expression of PP2A B56g3 reversed the tumorigencity of these cells. These findings suggest that PP2AB56g3 may play a tumor suppressor role, and alterations in expression of this subunit may contribute to cancer development. We aim to investigate the regulatory function of B56g3 subunit which may be involved in tumor suppressor function of PP2A. At present, we report that HA-tagged B56g3 is predominantly nuclear localized by GFP-tagging and immunofluorescence staining in both NIH 3T3 and COS-7 cells by transient transfection experiments. In addition, forced overexpression of B56g3 resulted in nuclear condensation in some transfected cells, and, to some extent, caused abnormal nuclear and cell morphology similar to apoptotic cells. Interestingly, in a NIH3T3 pool stably expressing B56g3, expression of B56g3 became evenly distributed throughout the cell in most of the cells. Moreover, overexpression of B56g3 retarded growth of NIH 3T3 cells, modestly decreased cells in G1, G2/M phase but increased in S phase of cell cycle. Besides, overexpression of B56g3 caused a significant increase in cell death of NIH 3T3 cells after serum withdrawl as compared to cells expressing vector alone. We also investigated whether B56g3 overexpression affected main signaling pathways regulating cell survival and proliferation in NIH 3T3 cells. And we found that overexpression of B56g3 resulted in significant decrease in phosphorylation of AKT at threonine 308 site and almost no changes of phosphorylation at serine473 site as compared to that of expressing vector-alone cells by serum stimulation . In addition, overexpression of B56g3 had little effect on phosphorylation of extracellular signal regulated kinases (Erks). In summary, our findings suggest that a role of PP2A B56g3 subunit in regulating cell cycle progression and apoptosis. In addition, B56g3 was predominantly localized to the nuclei in transiently transfected cells, while the expression of the protein became evenly distributed throughout the cells after stable expression. These results implicate that the subcellular localization of B56g3 may determine its regulatory function in cells.

目錄
誌謝
中文摘要......................................................................I
英文摘要....................................................................III
圖目錄........................................................................1
表目錄........................................................................2
A. 緒論.......................................................................3
一、蛋白質磷酸酶2A型 (Protein phosphatase 2A/ PP2A) ..........................3
二、PP2A的結構及次單元體......................................................3
三、各種B次單元體的介紹.......................................................4
四、各種B次單元體所調控的功能.................................................6
五、PP2A的調節功能 ...........................................................9
六、PP2A具有腫瘤抑制的功能...................................................12
七、PR61/B'r次單元扮演腫瘤抑制角色的重要證據.................................14
B. 研究方向..................................................................16
C. 材料與方法................................................................17
材料.........................................................................17
方法.........................................................................23
一、架構各種PP2A/B56r3次單元(subunit)的表現載體..............................23
(一)定序PP2A/B56r3次單元(subunit)之序列......................................23
(二)設計真核細胞及原核細胞的表現載體，以表現C端帶有一個HA tag B56r3HA次單元(subunit) ...................................................................23
(三)設計真核細胞表現載體，以表現一個N端帶有四個HA tag及C 端帶有EGFP螢光的4HAB56r3-EGFP的融合蛋白......................................................24
(四)設計Tetracycline-inducible-system的真核細胞表現載體......................24
(五)設計應用於反轉錄病毒感染系統的真核細胞表現載體，以表現一個N端帶有四個HA tag的4HAB56r3次單元(subunit) ...................................................25
(六)利用聚合酶連鎖反應 (Polymerase chain reaction)製造帶有合適限制酶切點及3’tag的產物....................................................................25
(七)膠體粹取 (Gel extraction)................................................27
(八)利用TA cloning的方法建構帶有PCR產物的pGEMT-T-easy載體...........................................................................27
(九)大腸桿菌轉形實驗 (E. Coli transformation) ...............................28
(十)藍白篩選 (blue and white screening) .....................................28
(十一)小量質體DNA的製備 ( mini prep) ........................................28
(十二)大量質體DNA的製備 ( maxi prep) ........................................29
二、細胞培養.................................................................30
(一)解凍細胞.................................................................30
(二)細胞繼代培養(subculture) ................................................31
(三)冷凍細胞.................................................................31
(四)細胞數目測定.............................................................31
(五)轉染 (Transfection) .....................................................32
(六)反轉錄病毒感染(Retroviral infection) ....................................33
(七)收集細胞.................................................................34
三、硫酸十二脂鈉聚丙烯醯胺凝膠蛋白質電泳法(SDS-PAGE)及西方點墨法 (Western Blotting) ...................................................................35
(一)蛋白質濃度的定量.........................................................35
(二)蛋白質樣品的製備 ........................................................35
(三)SDS-PAGE膠體電泳 ........................................................35
(四)西方點墨法(Western Blotting) ............................................36
四、利用免疫沉澱法(immunoprecipitation)偵測蛋白質的交互作用..................38
(一)製備Protein A/Protein B sepharose beads..................................38
(二)打破細胞取得蛋白質.......................................................38
(三) Pre-clearing cell lysate ...............................................38
(四)進行免疫沉澱法....................................... ...................39
五、利用免疫螢光染色法來偵測 B56r3 次單元在細胞中的表現位置..................39
(一)將細胞種植於24-well的細胞培養皿 .........................................39
(二)進行免疫螢光染色法.......................................................40
(三)利用倒立式螢光顯微鏡察.................................................. 41
六、利用PI染劑染DNA並進行流式細胞儀(FACS)分析................................41
(一)細胞的收集........................................... ...................41
(二) PI 染色.............................................. ..................41
(三)過濾細胞............................................. ...................42
(四)進行流式細胞儀(FACS)分析 ............................ ...................42
七、細胞生長曲線的分析.................................. ....................42
八、給予細胞 serum-withdrawl 的處理測試細胞的死亡數目..... ..................43
九、評估PP2A/B56r3HA對AKT473、AKT308及Erk1/2磷酸化的調節作用...........................................................................44
(一)給予細胞 serum withdrawl.................................................44
(二)加入serum 刺激AKT及Erk 活化..............................................44
(三)收集細胞並打破細胞取得蛋白質......................... ...................44
(一)SDS-PAGE及western-blot分析............................ ..................45
十、利用蛋白質體學的方法找出可能與PP2A/B56g3HA交互作用的蛋白...........................................................................45
(一)進行大量的免疫沉澱反應純化出與B56g3HA結合的蛋白複合物...........................................................................45
(二)進行一維的膠體電泳分離免疫沉澱的蛋白複合物 .......... ...................46
(三)進行銀染法找出比對照組多出的protein band .............. .................46
(四)找出比對照組多出的protein band進行質譜分析 ............ .................46
D. 實驗結果............................................. ....................48
一、設計表現B56r3次單元的各種表現載體........................................48
二、利用大腸桿菌表現系統表現並純化帶有六個Histidine His-B56r重組蛋白及帶有GST的GST-B56r3重組蛋白............................................................48
三、B56r3次單元在真核細胞中大部分集中在細胞核表現....... ....................49
四、 一些過度表現B56r3次單元的細胞會產生不正常的細胞核及細胞型態...........................................................................51
五、一些穩定表現B56r3次單元的NIH 3T3細胞群，顯示B56r3
單元在細胞核及細胞質都有表現.................................................52
六、過度表現B56r3次單元延遲NIH 3T3細胞的生長.................................53
七、過度表現B56r3HA次單元的NIH 3T3 細胞相較於表現
pMSCV vector 的細胞，經過 serum withdrawl 處理之後導致中
度的細胞死亡現象.............................................................53
八、 過度表現 B56r3HA 次單元的NIH3T3細胞可能使細胞停滯在S 時期而造成生長的遲緩現象.........................................................................54
九、過度表現B56r3HA 次單元導致AKT在Thr 308位點的磷
酸化減少；但是對於AKT Ser 473位點的磷酸化及Erk1/2的磷酸化則沒有顯著的影響...........................................................................55
十、免疫沉澱法證明B56r3HA 次單元能夠在NIH 3T3細胞中
PP2A2的A次單元及C次單元形成穩定複合物........................................57
十一、利用蛋白質體學的方法找到一個可能與B56r3HA 次單元交互作用的蛋白-Calcium-activated potassium channel..................................................58
E. 討論......................................................................60
F. 參考文獻 ............................................. ...................67
G. 圖........................................................................80
H. 表.......................................................................101
I. 附圖.....................................................................102
附圖一、PP2A的次單元體組成............................. ....................102
附圖二、各種不同的 B 次單元所扮演的細胞調節角色.............................103
附圖三、在細胞的發展過程中，PP2A 組成次單元的不正常表現、突變或缺失導致腫瘤的形成的分子轉機................................................................104
附圖四、ST 能夠取代 B56/PR61r次單元而與PP2A dimer結合，進而導致腫瘤的生成..........................................................................105
附圖五、pMSCV plasmid map...................................................106
附圖六、pCDNA3.1 plasmid map................................................107
附圖七、pEGFP-N1 plasmid map................................................108
附圖八、pRevTRE plasmid map.................................................109
附圖九、pCEP4 vector plasmid map............................................110
附圖十、pCDNA3.1HisC plasmid map............................................111
附圖十一、pGEM-T Easy plasmid map...........................................112
附圖十二、pGEX plasmid map..................................................113
附錄十三、pQE30 plasmid map.................................................114
J. 附表................................................. ...................115
一、B56 次單元家族各種異構型的不同細胞分布..................................115
二、PP2A 具有腫瘤抑制功能的證據.............................................116

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