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系統識別號 U0026-2908201401055100
論文名稱(中文) 探究TIP的細胞功能
論文名稱(英文) Investigate the cellular functions of type 2A-interacting protein (TIP)
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 102
學期 2
出版年 103
研究生(中文) 鍾承恩
研究生(英文) Cheng-En Chung
學號 T16014080
學位類別 碩士
語文別 英文
論文頁數 54頁
口試委員 指導教授-蔣輯武
口試委員-吳梨華
口試委員-陳玉玲
口試委員-黃暉升
中文關鍵字 蛋白質磷酸酶2A型  TIP  細胞運動 
英文關鍵字 PP2A  TIP  cell motility 
學科別分類
中文摘要 蛋白質磷酸酶 2A型 (PP2A)是真核細胞中一種主要的絲氨酸/蘇氨酸磷酸酶,參與調控多種細胞功能。典型的PP2A複合體是由一個結構次單元 (PP2A/A)及一個催化次單元 (PP2Ac)作為核心,再加上多變的調節次單元 (PP2A/B)所組成的三元複合體。然而除了典型的PP2A複合體,現已發現一些分子如Alpha4 (α4)、type 2A-interacting protein (TIP)會與PP2Ac產生交互作用而形成非典型PP2A複合體。為了更加了解TIP在細胞中的功能,我們在NIH3T3、HeLa、Hep3B及HepG2等細胞中,分別建立過度表現TIP或以核醣核酸干擾法將TIP表現減低的stable cell pools,並觀察TIP對細胞的形態特徵、生長特性、運動性及細胞爬行有什麼影響。我們發現在NIH3T3及HepG2細胞中過度表現TIP時,會增加細胞的增殖速度,但在HeLa細胞中過度表達TIP卻會減少細胞的增殖速度。接著,在HeLa細胞中以核醣核酸干擾法將TIP表現減低時,我們發現呈紡錘狀的細胞數目增多了,隨著細胞型態的改變,我們也發現當TIP過度表現於HeLa細胞中時,細胞的常態性運動及在傷痕癒合下的運動能力會下降。相反地,當以核醣核酸干擾法將TIP表現減低時則會增加細胞的常態性運動及在傷痕癒合的運動速度。接著,我們發現過度表達TIP造成actin網絡的增加,而將TIP表現減低時則會增加actin stress fiber的形成。接著,我們探討TIP可能是經由調控那些相關的訊息傳遞路徑來造成這些細胞現象,我們發現過度表現TIP僅造成PP2A B55調節次單元家族的表現量些微降低,相反的,卻可以提高B56γ3、α4及Akt的表現,但沒有影響PP2A/A及PP2Ac的表現量。用siRNA來降低TIP表現時,造成B56γ2、B56γ3、α4及Akt的表現量降低。我們進一步發現,過度表現TIP會使Akt的絲氨酸473位點的磷酸化增加,然而,過度表現TIP會減少激酶Akt的蘇氨酸308位點的磷酸化程度。此外,過度表現TIP會使mTOR下游的P70S6K的蘇氨酸389磷酸化減少,但卻對4EBP1的絲氨酸65的磷酸化沒有影響。總結以上,我們發現TIP在不同細胞中對細胞的增殖的調控不同,可以扮演促進也可以扮演抑制的角色。TIP會藉由控制actin stress fiber的形成來調控細胞形態特徵、細胞運動及細胞爬行。並且,TIP調控細胞現象可能是經由控制PP2A B55調節次單元家族、B56γ調節次單元家族、Akt、α4及P70S6K所參與的訊息傳遞路徑。
英文摘要 Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase involved in regulating a variety of cellular activities in mammalian cells. The canonical PP2A complex consists of a heterodimeric core enzyme, including a scaffolding subunit (A) and a catalytic subunit (PP2Ac), and a variable regulatory subunit (B). In addition to the canonical PP2A complex, several molecules, such as α4 and type 2A-interacting protein (TIP), have been identified to associate with PP2Ac to form non-canonical PP2A complexes. To understand more about the roles of TIP in cellular functions, we established pools of NIH3T3, HeLa, Hep3B, and HepG2 cells with stable TIP overexpression or knockdown of TIP and characterized these cells according to their morphological characteristics, growth properties, motility, and migration. We found that TIP overexpression increased proliferation of NIH3T3 cells and HepG2 cells, whereas TIP overexpression reduced proliferation of HeLa cells. HeLa cells with stable TIP knockdown showed increased numbers of cells with a spindle-shaped morphology. Consistent with the morphology change, TIP overexpression reduced cell motility and wound-healing migration in HeLa cells, whereas HeLa cells with stable TIP knockdown showed enhanced motility and migration. Concomitantly, TIP overexpression increased staining of actin web, while TIP knockdown increased amounts of actin stress fibers. Next, we investigated signaling pathways regulated by TIP. TIP overexpression slightly down-regulated the levels of the B55 family regulatory subunits of PP2A, whereas TIP overexpression increases protein levels of B56γ regulatory subunits, α4, and Akt in HeLa cells. Knockdown of TIP by siRNA reduced levels of B56γ2, B56γ3, α4, and Akt in HeLa cells. TIP overexpression increased protein levels of α4 and Akt, but not B56γ, in HepG2 cells. Conversely, modulating TIP levels did not change the levels of the A and C subunits. In addition, TIP overexpression increased phosphorylation levels of Akt at S473, whereas TIP overexpression decreased phosphorylation levels of Akt at T308 and phosphorylation levels of P70S6K at T389. TIP appears to affect P70S6K phosphorylation independent of mTORC1, since TIP overexpression did not affect phosphorylation levels of 4EBP1 at S65. In summary, our data demonstrate that TIP can either up-regulate or down-regulate cell proliferation, depending on cell types. TIP regulates morphology, cell motility, and migration through modulating actin filament rearrangements. Taken together, TIP-regulated cellular phenotypes may be attributable to modulating signaling pathways involving PP2A B55 family subunits, B56γ family subunits, Akt, α4, and P70S6K.
論文目次 中文摘要 I
Abstract III
誌謝 V
List of Contents VI
List of Figures VIII
List of Abbreviations IX
Introduction 1
Protein phosphatase 2A (PP2A) 2
Non-canonical PP2A complexes 2
Type 2A-interacting protein (TIP) 3
PP2A regulates cell morphogenesis and cell motility 4
PP2A regulates Akt and mTOR pathways 6
Hypothesis 8
Specific aims 8
Objective 8
Materials and Methods 9
Antibodies and reagents 10
Reagents for DNA cloning 11
Cell culture, retro/lenti viral preparation, and cell lines 11
DNA constructs 13
Migration assay 13
Wound healing assay 14
Immunofluorescence staining and microscopy 14
Western blotting 15
Cell proliferation assay 15
Results 17
TIP modulates cell morphology and regulates stress fiber formation 18
TIP reduces cell motility and migration 19
TIP regulates cell proliferation 20
Signaling pathways regulated by TIP 20
Conclusion 22
Discussion 23
TIP regulates cell motility 24
TIP regulates cell proliferation in a cell-type dependent manner 25
The inconsistent outcomes between TIP overexpression and TIP knockdown 26
References 29
Figures 35
Appendix 52
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