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系統識別號 U0026-0407201215095600
論文名稱(中文) 探討磷酸酶PP2A核質穿梭的機制及功能性角色
論文名稱(英文) Mechanisms and functional roles of nuclear-cytoplasmic shuttling of protein phosphatase 2A
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 100
學期 2
出版年 101
研究生(中文) 李定遠
研究生(英文) Ting-Yuan Lee
學號 S58951536
學位類別 博士
語文別 英文
論文頁數 122頁
口試委員 指導教授-蔣輯武
召集委員-張南山
口試委員-林以行
口試委員-楊倍昌
口試委員-徐士蘭
口試委員-孟子青
中文關鍵字 磷酸酶 PP2A  亞細胞分佈特性  B56γ3  細胞週期  核質穿梭 
英文關鍵字 PP2A  subcellular localization  B56γ3  cell cycle  nuclear-cytoplasmic shuttling 
學科別分類
中文摘要 磷酸酶 Protein phosphatase 2A (PP2A),屬於一種絲氨酸與蘇氨酸磷酸酶,廣泛表現真核生物細胞中,而且參與了細胞內許多的調節作用;例如:細胞的訊息傳遞(Signal transduction)、細胞週期的進行(Cell cycle progression)、細胞的生長(Cell growth)以及參與細胞凋亡(Apoptosis)的進行等,而當PP2A功能失常時,則會導致正常細胞的病變。PP2A的組成為三元複合體,主要由一個結構次單元,稱之為A次單元;及一個催化次單元,稱之為C次單元;以及高多樣性且具有調節功能的B次單元所組成。目前普遍認為PP2A的受質特異性(Substrate specificity)與亞細胞分佈特性(Subcellular localization)是由B次單元所調控。而我們的研究則主要是探討B56γ3調節次單元對於PP2A次細胞分佈的調控機制與其不同分佈位置對於細胞功能的影響。我們證實B56γ3的分佈是多變的,在恆穩態與細胞週期G1期的細胞中,B56γ3大致呈現核質均勻分佈(Homogenous);然而隨著細胞週期進入G1與S期臨界期與S期時,B56γ3則明顯集中表現於細胞核內,同時在核內的PP2A/AC dimer也隨之增加。另一方面,大量表現B56γ3於細胞核內時,亦能提升PP2A/AC dimer於細胞核內的分佈,且細胞核內PP2A活性也相對來得高。但當抑制內生性B56γ2與B56γ3的表現時,則可顯著降低PP2A於細胞核內的分佈與活性。在NIH3T3細胞中,大量表現B56γ3可降低p27在蘇氨酸187上的磷酸化,並提高p27蛋白質分子的表現來延緩細胞的生長。當抑制HeLa細胞內內生性B56γ3的表現後,則會造成p27蛋白質的降解因而加速細胞生長。接著,我們也持續研究B56γ3的核運送機制,我們發現核運送受體蛋白-Importin α與Importin β會分別直接與B56γ3上的氨基酸306到405這一段區域結合。且當B56γ3上的絲胺酸440被突變成持續磷酸化時,可顯著增加B56γ3進入細胞核的情形,並減緩細胞的生長。但當絲胺酸440被突變成持續去磷酸化時,則會降低B56γ3於細胞核內的表現與影響PP2A B56γ3對於細胞生長調控的能力。此外,絲胺酸440上的磷酸化也會隨著細胞週期的進行而有所改變,進而影響B56γ3蛋白的穩定性。結論,我們已證實PP2A的調控次單元-B56γ3其核質穿梭的機制是依細胞週期而進行的,並藉由調控細胞週期調控因子,如p27,來調控細胞週期的進行。再者,B56γ3調節次單元可能是以透過“非典型的NLS”序列來調控進入細胞核,經由Importin α-dependent與Importin α-independent的機制來被送入細胞核內。
英文摘要 Protein phosphatase 2A (PP2A) is one major serine/threonine protein phosphatase in eukaryotic cells that has a multitude of functions inside the cell, acting through various targets in cell signal pathways. PP2A consists of a catalytic subunit (PP2A/C) which forms a stable complex with the scaffold subunit (PP2A/A), and this heterodimer (PP2A/AC) associates with regulatory proteins, termed regulatory subunits, to form trimeric holoenzymes attributed with distinct substrate specificity and targeted to different subcellular compartments. In this study, we demonstrated that the subcellular localization of the regulatory subunit B56γ3 is regulated in a cell cycle-dependent manner. Notably, B56γ3 is most concentrated in the nucleus at the G1/S interface and S phase. The S phase-specific nuclear enrichment of B56γ3 was accompanied by an increment of nuclear PP2A/AC dimer and also by nuclear PP2A activity. Overexpression of B56γ3 promoted the nuclear accumulation of PP2A/AC, whereas silencing both B56γ2 and B56γ3 blocked the S phase-specific nuclear accumulation of PP2A/AC and S phase-specific increases in the nuclear phosphatase activity of PP2A. In NIH3T3 cells, overexpressing B56γ3 reduced the phosphorylation of p27 at Thr187, and the protein level of p27 was concomitantly elevated, leading to a delay in the G1 to S transition and to retarded cell proliferation. Consistently, when endogenous B56γ3 expression was knocked down in HeLa cells, p27 protein expression was reduced, alone with increased cell proliferation. Further, to determine the nuclear transport, B56γ3 was shown to directly interact with nuclear transport receptors Importin α and β via a tentative nuclear targeting sequence (amino acid 306-405). B56γ3 was phosphorylated at residue Ser440, and a phosphorylation mimetic mutant (S440D) showed significantly increased nuclear localization of B56γ3 and inhibition of cell proliferation. Conversely, the dephosphorylation defective mutant of Ser440 (S440A) reduced the nuclear accumulation of B56γ3 and impaired the inhibitory effect on cell proliferation and the retardation of cell growth. In addition, the B56γ3 protein levels varied during the course of cell cycle progression, and its phosphorylation of Ser440 played a critical role in stabilizing B56γ3. In conclusion, our data demonstrates that the B56γ3-regulatory subunit of PP2A shuttles between the nucleus and cytoplasm in a cell cycle-dependent manner to regulate cell cycle progression with the participation of p27, a cell-cycle controller. Furthermore, these results suggest that the B56γ3 regulatory subunit may enter nucleus through a non-classical NLS-mediated and both Importin α-dependent and Importin α-independent mechanism.
論文目次 中文摘要 Ⅰ
Abstract Ⅱ
誌謝 Ⅳ
Contents Ⅴ
Figure Contents Ⅷ
Appendix Contents Ⅹ
Abbreviations list Ⅺ
Introduction 1
Protein Phosphatase 2A (PP2A) 2
The multimeric structure of PP2A 3
The regulatory B subunits 4
The biological roles of PP2A 6
The subcellular localization of PP2A holoenzyme 9
The B56γ family 11
The functions of PP2A as a tumor suppressor 12
The regulatory subunit B56γ3 13
The regulation of nuclear-cytoplasmic shuttling mechanism 14
Nuclear protein import mechanism 15
The dynamic localization of PP2A 16
The post translation modification of PP2A 17
Research Motivation 18
Materials and Methods 21
Antibodies and Reagents 22
Cell Culture, Cell Line and Transfection 22
Selection of Cells stably Expressing HA-tagged B56γ3, B56γ3 shRNA or Vector only 23
Cell Cycle Synchronize 23
Cell Cycle Analysis 24
Western Blotting and Co-immunoprecipitation 24
Subcellular Fractionation 25
Immunofluorescence and Microscopy 25
Measurement of the PP2A Activity 26
Recombination Protein Preparation 26
In Vitro Pulldown Analysis 27
OptiPrep Density Gradient Centrifugation 27
Results 29
The subcellular distribution of the B56γ3 subunit is regulated in a cell cycle-dependent manner 30
B56γ3 overexpression increases nuclear localization of the PP2A/A and PP2A/C subunits of PP2A 32
Biochemical fraction reveal S phase-specific increase in nuclear distribution of B56γ3, PP2A/A and PP2A/C subunits 34
The B56γ subunits regulate nuclear PP2A activity that is increased in an S phase-specific manner 37
B56γ3 regulates the G1 to S transition of the cell cycle and regulates p27KIP1 levels 38
Lack the potential NLS sequence does not affect the nuclear accumulation of B56γ3 42
Ser440-phosphorylation within the linker segment of B56γ3 is critical for S-phase nuclear accumulation 43
The total protein expression and phosphorylated level (S440) of B56γ3 are changed with cell cycle progression 44
The phosphorylation at Ser440 whether or not will affect the protein stability of B56γ3 45
Identify the Importin protein interactive sequence of B56γ3 46
Identification of segments within a.a. 306-405 which to affect Importin α or Importin β binds to B56γ3 47
Phosphorylation defective mutant at Ser440 of B56γ3 results in the reduction of interaction Importin α and β 48
Conclusion & Discussion 50
The B56γ3 subunit shuttles between the nucleus and cytoplasm in a cell-cycle dependent manner 51
B56γ3 overexpression increases the nuclear localization of A and C subunits 52
The nuclear PP2A activity is increased by B56γ3 in an S phase-dependent manner 53
PP2A AB56γ3C monitors the cell proliferation through regulating the cell cycle progression 54
The B56γ3-containing PP2A holoenzyme regulates the G1 to S transition, by modulating p27 protein level 55
Regulation of nuclear localization of B56γ3 57
Phosphorylation of B56γ3 at Ser440 regulates the protein stability 59
References 61
Figur 73
Appendix 112
Curriculum Vitae 122
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