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系統識別號 U0026-2708201323011000
論文名稱(中文) 探討腺苷單磷酸活化激酶調節磷酸酶PP2A調節次單元B56γ3細胞分佈及腫瘤抑制的角色
論文名稱(英文) Investigate the role of AMPK in regulating the subcellular localization and tumor suppressor activity of the B56γ3 regulatory subunit of protein phosphatase 2A
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 101
學期 2
出版年 102
研究生(中文) 高岱瑩
研究生(英文) Tai-Ying Kao
學號 T16004051
學位類別 碩士
語文別 英文
論文頁數 63頁
口試委員 指導教授-蔣輯武
口試委員-張南山
口試委員-凌斌
口試委員-洪良宜
中文關鍵字 蛋白質磷酸酶2A型  腺苷單磷酸活化激酶 
英文關鍵字 PP2A  AMPK 
學科別分類
中文摘要 磷酸酶PP2A是一種絲氨酸/蘇氨酸磷酸酶,組成PP2A的三個次單元分別是構成結構骨架的A次單元,負責催化功能的C次單元,以及種類相當多樣的調節性B次單元。這些種類繁多的B次單元可以決定PP2A完全酶的活性、受質特異性,以及其在細胞中的位置。腺苷單磷酸活化蛋白激酶AMPK是一個感應細胞內能量代謝的關鍵調控者,在壓力之下會被活化,例如營養缺乏,可以調控細胞週期停滯在G1期。同樣的,PP2A的B56γ3調節性次單元也參與調控細胞週期,大量表現B56γ3會延遲細胞從G1進展進入到S期。先前我們研究發現B56γ3上的一段區域 (aa 413 到 aa 461) 會影響B56γ3座落在核的分佈,經由序列分析預測在這段區域上的絲氨酸440是AMPK可能磷酸化的位點。免疫沉澱實驗證明了B56γ3會和AMPKα1與持續性活化態AMPKα1(1-312)兩者有交互作用,大量表現持續性活化態AMPKα1(1-312)或AMPKα1β1γ1複合體在葡萄糖缺少情況下會提高絲氨酸440磷酸化。進一步我們發現處理AMPK的抑制劑compound C會降低絲氨酸440磷酸化和B56γ3在核的分佈,而處理AMPK活化劑AICAR會提高絲氨酸440磷酸化和B56γ3在核的分佈。利用siRNA降低AMPKα1表現,發現其的確降低了絲氨酸440磷酸化和B56γ3在核的分佈。此外,在葡萄糖缺少情況下,大量表現AMPKα1會提高絲氨酸440磷酸化和B56γ3在核的分佈。但是當大量表現持續性活化態AMPKα1(1-312)並沒有顯著影響到B56γ3在核的分佈。此外,我們發現抑制AMPK時不只降低了絲氨酸440磷酸化,同時也會降低了B56γ3的蛋白質量。最後,我們用compound C處理穩定表現B56γ3或vector的NIH3T3細胞來抑制AMPK,結果我們發現抑制AMPK能夠降低穩定表現B56γ3下對於在緩解接觸抑制後的p27蛋白質量的提高作用。
英文摘要 Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase which comprises three subunits, including a scaffolding A subunit, a catalytic C subunit, and a variable regulatory B subunit. The B subunits are involved in determining the activity, substrate specificity, and subcellular localization of PP2A. The AMP-activated protein kinase (AMPK) plays a pivotal role in regulating cellular metabolism and is activated upon stress, including nutrient deprivation, to arrest cell cycle at G1 phase. Similarly, the B56γ3 regulatory subunit of PP2A is involved in regulating cell cycle at the G1/S transition and overexpression of B56γ3 delays the G1/S transition. We have previously shown that a domain encompassing aa 413 to aa 461 is critical in regulating nuclear localization of B56γ3, and sequence analysis predicted Ser440, residing within this nuclear localization regulating domain, as a potential phosphorylation site of AMPK. In this study, we show that B56γ3 interacts with both the AMPKα1 and the constitutively active form of AMPKα1 (AMPKα1CA or AMPKα1 (1-312)) by co-immunoprecipitation, and co-expression of the constitutively active form of AMPKα1 or heterotrimeric AMPKα1β1γ1 complexes under glucose starvation increases Ser440 phosphorylation of B56γ3. Treatment of cells with AMPK inhibitor compound C reduces Ser440 phosphorylation levels and nuclear localization of B56γ3, whereas treatment of cells with AMPK activator AICAR increases Ser440 phosphorylation levels and nuclear localization of B56γ3 in a dose-dependent manner. Further, siRNA knockdown of AMPKα1 reduces both phosphorylation levels of Ser440 and the nuclear localization of B56γ3, whereas AMPKα1 overexpression increases Ser440 phosphorylation and nuclear localization of B56γ3 under glucose starvation. Unexpectedly, overexpression of AMPKα1CA (1-312) does not significantly affect the nuclear localization of B56γ3. Further, we found that AMPK inhibition not only affects Ser440 phosphorylation of B56γ3, but also reduces the total protein level of B56γ3. Lastly, inhibition of AMPK by compound C treatment blocks the augmenting effects of B56γ3 overexpression on the p27 protein levels after cells were released from contact inhibition.
In summary, our data demonstrate that AMPK regulates Ser440 phosphorylation to control nuclear localization and stability of B56γ3, and may regulate B56γ3’s tumor suppressor activity through regulation of p27.
論文目次 摘 要 I
Abstract II
Table of Contents 1
List of Figures 3
List of Abbreviations 4
Introduction 5
Protein Phosphatase 2A 6
The structure of PP2A 6
B regulatory subunits of PP2A 7
PP2A functions as a tumor suppressor 8
5’-AMP-activated protein kinase (AMPK) 9
The Nuclear Import Pathway 11
Objectives 12
Materials and Methods 13
Antibodies 14
Reagents 14
DNA constructs 15
Cell culture 16
Contact inhibition 16
Knockdown of AMPKα1 by siRNA 17
Virus preparation and selection of cells stably expressing HA-tagged B56γ3 17
Immunofluorescence 18
Western Blotting 18
Immunoprecipitation 19
Results 20
The phosphorylation level of Ser440 is decreased by compound C treatment and increased by AICAR treatment in a dose-dependent manner 21
PP2A-mediated AMPK inhibition decreases the phosphorylation levels of Ser440 22
Knockdown of AMPKα1 by siRNA decreases the phosphorylation level of Ser440 22
AMPKα1 associates with B56γ3 and increases Ser440 phosphorylation of B56γ3 23
AMPKα regulates nuclear localization of B56γ3 24
Ser440 phosphorylation of B56γ3 is increased under glucose starvation 25
AMPK increases nuclear localization of B56γ3 under glucose starvation 26
Investigate the role of AMPK in B56γ3-mediated increases of p27 protein levels 26
Conclusion 27
Discussion 28
The role of PP2A in the regulation of AMPK phosphorylation and activity 29
AMPK phosphorylates B56γ3 at Ser440 29
The possible mechanisms underlying AMPK regulates B56γ3 30
Both AMPK and B56γ3 regulate cell cycle regulator p27 to control cell proliferation 31
Phosphorylatetion of B56γ3 at Ser440 by AMPK regulates B56γ3 protein stability 32
References 33
Figures 41
Appendix 61
作者簡歷 63
參考文獻 1.Shi Y (2009) Serine/threonine phosphatases: mechanism through structure. Cell 139(3):468-484.
2.Janssens V & Goris J (2001) Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. The Biochemical journal 353(Pt 3):417-439.
3.Lechward K, Awotunde OS, Swiatek W, & Muszynska G (2001) Protein phosphatase 2A: variety of forms and diversity of functions. Acta biochimica Polonica 48(4):921-933.
4.Chen YG, et al. (2006) Activin signaling and its role in regulation of cell proliferation, apoptosis, and carcinogenesis. Experimental biology and medicine 231(5):534-544.
5.Cho US & Xu W (2007) Crystal structure of a protein phosphatase 2A heterotrimeric holoenzyme. Nature 445(7123):53-57.
6.Eichhorn PJ, Creyghton MP, & Bernards R (2009) Protein phosphatase 2A regulatory subunits and cancer. Biochimica et biophysica acta 1795(1):1-15.
7.Groves MR, Hanlon N, Turowski P, Hemmings BA, & Barford D (1999) The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs. Cell 96(1):99-110.
8.Ruediger R, et al. (1992) Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus. Molecular and cellular biology 12(11):4872-4882.
9.Ruediger R, Hentz M, Fait J, Mumby M, & Walter G (1994) Molecular model of the A subunit of protein phosphatase 2A: interaction with other subunits and tumor antigens. Journal of virology 68(1):123-129.
10.Xu Y, et al. (2006) Structure of the protein phosphatase 2A holoenzyme. Cell 127(6):1239-1251.
11.Janssens V, Longin S, & Goris J (2008) PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail). Trends in biochemical sciences 33(3):113-121.
12.Gorlich D, et al. (1995) Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope. Current biology : CB 5(4):383-392.
13.McCright B & Virshup DM (1995) Identification of a new family of protein phosphatase 2A regulatory subunits. The Journal of biological chemistry 270(44):26123-26128.
14.Seeling JM, et al. (1999) Regulation of beta-catenin signaling by the B56 subunit of protein phosphatase 2A. Science 283(5410):2089-2091.
15.Ito A, et al. (2000) A truncated isoform of the PP2A B56 subunit promotes cell motility through paxillin phosphorylation. The EMBO journal 19(4):562-571.
16.Janssens V, Goris J, & Van Hoof C (2005) PP2A: the expected tumor suppressor. Current opinion in genetics & development 15(1):34-41.
17.Letourneux C, Rocher G, & Porteu F (2006) B56-containing PP2A dephosphorylate ERK and their activity is controlled by the early gene IEX-1 and ERK. The EMBO journal 25(4):727-738.
18.McCright B, Rivers AM, Audlin S, & Virshup DM (1996) The B56 family of protein phosphatase 2A (PP2A) regulatory subunits encodes differentiation-induced phosphoproteins that target PP2A to both nucleus and cytoplasm. The Journal of biological chemistry 271(36):22081-22089.
19.Bode AM & Dong Z (2004) Post-translational modification of p53 in tumorigenesis. Nature reviews. Cancer 4(10):793-805.
20.Gigena MS, Ito A, Nojima H, & Rogers TB (2005) A B56 regulatory subunit of protein phosphatase 2A localizes to nuclear speckles in cardiomyocytes. American journal of physiology. Heart and circulatory physiology 289(1):H285-294.
21.Flegg CP, et al. (2010) Nuclear export and centrosome targeting of the protein phosphatase 2A subunit B56alpha: role of B56alpha in nuclear export of the catalytic subunit. The Journal of biological chemistry 285(24):18144-18154.
22.Lee TY, et al. (2010) The B56gamma3 regulatory subunit of protein phosphatase 2A (PP2A) regulates S phase-specific nuclear accumulation of PP2A and the G1 to S transition. The Journal of biological chemistry 285(28):21567-21580.
23.Bialojan C & Takai A (1988) Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. The Biochemical journal 256(1):283-290.
24.Schonthal AH (2001) Role of serine/threonine protein phosphatase 2A in cancer. Cancer letters 170(1):1-13.
25.Calin GA, et al. (2000) Low frequency of alterations of the alpha (PPP2R1A) and beta (PPP2R1B) isoforms of the subunit A of the serine-threonine phosphatase 2A in human neoplasms. Oncogene 19(9):1191-1195.
26.Colella S, et al. (2001) Reduced expression of the Aalpha subunit of protein phosphatase 2A in human gliomas in the absence of mutations in the Aalpha and Abeta subunit genes. International journal of cancer. Journal international du cancer 3(6):798-804.
27.Suzuki K & Takahashi K (2003) Reduced expression of the regulatory A subunit of serine/threonine protein phosphatase 2A in human breast cancer MCF-7 cells. International journal of oncology 23(5):1263-1268.
28.Wang SS, et al. (1998) Alterations of the PPP2R1B gene in human lung and colon cancer. Science 282(5387):284-287.
29.Takagi Y, et al. (2000) Alterations of the PPP2R1B gene located at 11q23 in human colorectal cancers. Gut 47(2):268-271.
30.Ruediger R, Pham HT, & Walter G (2001) Alterations in protein phosphatase 2A subunit interaction in human carcinomas of the lung and colon with mutations in the A beta subunit gene. Oncogene 20(15):1892-1899.
31.Pallas DC, et al. (1990) Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A. Cell 60(1):167-176.
32.Kamibayashi C, et al. (1994) Comparison of heterotrimeric protein phosphatase 2A containing different B subunits. The Journal of biological chemistry 269(31):20139-20148.
33.Cayla X, Ballmer-Hofer K, Merlevede W, & Goris J (1993) Phosphatase 2A associated with polyomavirus small-T or middle-T antigen is an okadaic acid-sensitive tyrosyl phosphatase. European journal of biochemistry / FEBS 214(1):281-286.
34.Chen W, et al. (2004) Identification of specific PP2A complexes involved in human cell transformation. Cancer cell 5(2):127-136.
35.Deichmann M, Polychronidis M, Wacker J, Thome M, & Naher H (2001) The protein phosphatase 2A subunit Bgamma gene is identified to be differentially expressed in malignant melanomas by subtractive suppression hybridization. Melanoma research 11(6):577-585.
36.Hardie DG, Carling D, & Carlson M (1998) The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annual review of biochemistry 67:821-855.
37.Dyck JR, et al. (1996) Regulation of 5'-AMP-activated protein kinase activity by the noncatalytic beta and gamma subunits. The Journal of biological chemistry 271(30):17798-17803.
38.Kemp BE, et al. (1999) Dealing with energy demand: the AMP-activated protein kinase. Trends in biochemical sciences 24(1):22-25.
39.Steinberg GR & Kemp BE (2009) AMPK in Health and Disease. Physiological reviews 89(3):1025-1078.
40.Adamo HP & Grimaldi ME (1998) Functional consequences of relocating the C-terminal calmodulin-binding autoinhibitory domains of the plasma membrane Ca2+ pump near the N-terminus. The Biochemical journal 331 ( Pt 3):763-766.
41.Warden SM, et al. (2001) Post-translational modifications of the beta-1 subunit of AMP-activated protein kinase affect enzyme activity and cellular localization. The Biochemical journal 354(Pt 2):275-283.
42.Mitchelhill KI, et al. (1997) Posttranslational modifications of the 5'-AMP-activated protein kinase beta1 subunit. The Journal of biological chemistry 272(39):24475-24479.
43.Polekhina G, et al. (2003) AMPK beta subunit targets metabolic stress sensing to glycogen. Current biology : CB 13(10):867-871.
44.Hardie DG & Hawley SA (2001) AMP-activated protein kinase: the energy charge hypothesis revisited. BioEssays : news and reviews in molecular, cellular and developmental biology 23(12):1112-1119.
45.Minokoshi Y, et al. (2002) Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase. Nature 415(6869):339-343.
46.Jones RG, et al. (2005) AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Molecular cell 18(3):283-293.
47.Short JD, et al. (2008) AMP-activated protein kinase signaling results in cytoplasmic sequestration of p27. Cancer research 68(16):6496-6506.
48.Short JD, et al. (2010) AMPK-mediated phosphorylation of murine p27 at T197 promotes binding of 14-3-3 proteins and increases p27 stability. Molecular carcinogenesis 49(5):429-439.
49.Hardie DG, Ross FA, & Hawley SA (2012) AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nature reviews. Molecular cell biology 13(4):251-262.
50.Hardie DG (2004) The AMP-activated protein kinase pathway--new players upstream and downstream. J Cell Sci 117(Pt 23):5479-5487.
51.Harreman MT, et al. (2004) Regulation of nuclear import by phosphorylation adjacent to nuclear localization signals. The Journal of biological chemistry 279(20):20613-20621.
52.Gorlich D & Kutay U (1999) Transport between the cell nucleus and the cytoplasm. Annual review of cell and developmental biology 15:607-660.
53.Harel A & Forbes DJ (2004) Importin beta: conducting a much larger cellular symphony. Molecular cell 16(3):319-330.
54.Rout MP & Aitchison JD (2001) The nuclear pore complex as a transport machine. The Journal of biological chemistry 276(20):16593-16596.
55.Poon IK & Jans DA (2005) Regulation of nuclear transport: central role in development and transformation? Traffic 6(3):173-186.
56.Fried H & Kutay U (2003) Nucleocytoplasmic transport: taking an inventory. Cellular and molecular life sciences : CMLS 60(8):1659-1688.
57.Efthymiadis A (1998) The HIV-1 Tat Nuclear Localization Sequence Confers Novel Nuclear Import Properties. Journal of Biological Chemistry 273(3):1623-1628.
58.Xiao CY (1997) SV40 Large Tumor Antigen Nuclear Import Is Regulated by the Double-stranded DNA-dependent Protein Kinase Site (Serine 120) Flanking the Nuclear Localization Sequence. Journal of Biological Chemistry 272(35):22191-22198.
59.Pouton CW, Wagstaff KM, Roth DM, Moseley GW, & Jans DA (2007) Targeted delivery to the nucleus. Advanced drug delivery reviews 59(8):698-717.
60.Xue Y, et al. (2008) GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy. Molecular & cellular proteomics : MCP 7(9):1598-1608.
61.Blom N, Gammeltoft S, & Brunak S (1999) Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. Journal of molecular biology 294(5):1351-1362.
62.Wang MY & Unger RH (2005) Role of PP2C in cardiac lipid accumulation in obese rodents and its prevention by troglitazone. American journal of physiology. Endocrinology and metabolism 288(1):E216-221.
63.Wu Y, Song P, Xu J, Zhang M, & Zou MH (2007) Activation of protein phosphatase 2A by palmitate inhibits AMP-activated protein kinase. The Journal of biological chemistry 282(13):9777-9788.
64.Kim KY, et al. (2009) Adiponectin-activated AMPK stimulates dephosphorylation of AKT through protein phosphatase 2A activation. Cancer research 69(9):4018-4026.
65.Kodiha M, Rassi JG, Brown CM, & Stochaj U (2007) Localization of AMP kinase is regulated by stress, cell density, and signaling through the MEK-->ERK1/2 pathway. American journal of physiology. Cell physiology 293(5):C1427-1436.
66.Jager S, Handschin C, St-Pierre J, & Spiegelman BM (2007) AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha. Proceedings of the National Academy of Sciences of the United States of America 104(29):12017-12022.
67.Hardie DG (2011) AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function. Genes & development 25(18):1895-1908.
68.Oakhill JS, et al. (2010) beta-Subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by AMP-activated protein kinase (AMPK). Proceedings of the National Academy of Sciences of the United States of America 107(45):19237-19241.
69.McBride A, Ghilagaber S, Nikolaev A, & Hardie DG (2009) The glycogen-binding domain on the AMPK beta subunit allows the kinase to act as a glycogen sensor. Cell metabolism 9(1):23-34.
70.Vlach J, Hennecke S, & Amati B (1997) Phosphorylation-dependent degradation of the cyclin-dependent kinase inhibitor p27. The EMBO journal 16(17):5334-5344.
71.Short KM, Hopwood B, Yi Z, & Cox TC (2002) MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders. BMC cell biology 3:1.
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