進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-0812200915253362
論文名稱(中文) STIM1在細胞分裂的蛋白質修飾
論文名稱(英文) The Modification of STIM1 During Mitosis
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 97
學期 2
出版年 98
研究生(中文) 詹舒蘋
研究生(英文) Shu-Ping Chan
電子信箱 s2696101@mail.ncku.edu.tw
學號 s2696101
學位類別 碩士
語文別 中文
論文頁數 64頁
口試委員 口試委員-張偉嶠
指導教授-陳炳焜
指導教授-張文昌
中文關鍵字 內質網  鈣離子 
英文關鍵字 STIM1  calcium 
學科別分類
中文摘要 近幾年來stromal interaction molecule 1(STIM1)被認為是一個位於內質網(endoplasmic reticulum)膜上的鈣離子偵測器,調節細胞內鈣離子濃度[1]。然而現今對於STIM1是否參與細胞移動、細胞增生以及細胞凋亡並不清楚。為了研究STIM1的活化與細胞增生之間的關係,我們利用siRNA技術壓制細胞內STIM1蛋白質表現量,結果顯示在STIM1蛋白質表現量較低的組別中,細胞增生能力受到抑制。進一步想了解細胞增生能力受到抑制是不是因為細胞週期受到影響而無法順利進行。我們發現STIM1在細胞分裂時期(Mitosis phase)蛋白質量不僅明顯下降並且也受到磷酸化修飾。除此之外,當我們處理廣為人知的STIM1活化劑thapsigargin (TG)時並沒有觀察到相同的現象,了解STIM1的蛋白質修飾可能與其活性無關。然而當細胞處理proteasome抑制劑MG132後發現mitosis phase出現未磷酸化態的STIM1。在過度表現STIM1的細胞中會促進COX2 promoter活性增加,並且STIM1 siRNA會抑制EGF所誘導的COX2 promoter活性。Calyculin A為protein phosphatase 2A (PP2A)及protein phosphatase 1 (PP1)的抑制劑,能夠保持STIM1維持在磷酸化態。綜合以上實驗結果得知STIM1在mitosis phase受到磷酸化修飾,這樣的修飾可能跟蛋白質安定性相關,又STIM1能夠影響COX2基因表現。未來我們將進一步找出磷酸酯酶(phosphatase)並釐清磷酸化態的STIM1在細胞內的生理功能。
英文摘要 Stromal interaction molecule 1(STIM1)has recently been identified as a endoplasmic reticulum(ER)Ca2+ sensor for store-operated Ca2+ entry. When STIM1 senses ER luminal Ca2+ depletion, it will redistribute from ER membrane to the plasma membrane to trigger store-operated calcium entry(SOCE), resulting in causing Ca2+ influx [1]. STIM1 interacts with store-operated calcium channels by C-terminal domains. However, little is known how STIM1 translocates to cell membrane. Since STIM1 could regulate Ca2+ concentration that suggests it may be also involved in cell migration, cell proliferation and apoptosis. In order to study the correlation between the activation of STIM1 and cell proliferation, the cell growth was measured in STIM1 knockdown cells. Our previous results showed that STIM1 siRNA significantly inhibited cell proliferation. We also checked whether STIM1 siRNA-inhibited cell growth was mediated by cell cycle regulation. Interestingly, we found that STIM1 was phosphorylated when cells were in M phase. Furthermore thapsigargin which is a well-known activator of STIM1 had no effect on the phosphorylation of STIM1. It seems that there is no correlation between the modification and protein activity of STIM1. When cells were treated with MG132 which is a proteasome inhibitor , we found that the non-phosphorylated STIM1 was increased in mitosis phase. In addition, COX2 promoter activity was enhanced in STIM1-overexpressed cells. STIM1 siRNA also inhibited EGF-induced COX2 promoter activity. Calyculin A which is a PP2A and PP1 inhibitor maintained the phosphorylation of STIM1. In conclusion we discoveried that STIM1 is phosphrylated in mitosis phase and this modification may be related to ptrotein stabiblity. In the future we will want to find out the phosphatase
and clarify the cellular function of phosphorylated STIM1.
論文目次 中文摘要-----------------------------------------2
英文摘要-----------------------------------------3
附圖目錄-----------------------------------------6
縮寫指引-----------------------------------------7
第一章 緒論--------------------------------------8
第二章 實驗材料及方法
來源---------------------------------------------14
實驗材料及方法-----------------------------------20
第三章 實驗結果
第一節 STIM1與cell proliferation-----------------36
第二節 STIM1與cell cycle-------------------------37
(一) STIM1在mitosis phase受到後轉譯修飾
(二) STIM1後轉譯修飾與細胞型態的關係
(三) STIM1後轉譯修飾與nocodazole藥物副作用的關係
第三節 STIM1的後轉譯修飾-------------------------39
(一) STIM1在mitosis phase 受到磷酸化修飾
(二) STIM1的去磷酸化
第四節 磷酸化態STIM1在細胞內可能扮演的生理角色---41
(一) 磷酸化態STIM1與其活性的關係
(二) 磷酸化態STIM1與其蛋白質安定性的關係
(三) STIM1與cyclooxygenase 2(COX2)的基因表現
第四章 討論--------------------------------------44
第五章 參考文獻----------------------------------49
附圖-------------------------------------------- 53
參考文獻 1. Zhang SL, Yu Y, Roos J, Kozak JA, Deerinck TJ, Ellisman MH, Stauderman KA, Cahalan MD: STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature 2005, 437(7060):902-905.
2. Reid LH, West A, Gioeli DG, Phillips KK, Kelleher KF, Araujo D, Stanbridge EJ, Dowdy SF, Gerhard DS, Weissman BE: Localization of a tumor suppressor gene in 11p15.5 using the G401 Wilms' tumor assay. Hum Mol Genet 1996, 5(2):239-247.
3. Sabbioni S, Barbanti-Brodano G, Croce CM, Negrini M: GOK: a gene at 11p15 involved in rhabdomyosarcoma and rhabdoid tumor development. Cancer Res 1997, 57(20):4493-4497.
4. Spassova MA, Soboloff J, He LP, Xu W, Dziadek MA, Gill DL: STIM1 has a plasma membrane role in the activation of store-operated Ca(2+) channels. Proc Natl Acad Sci U S A 2006, 103(11):4040-4045.
5. Manji SS, Parker NJ, Williams RT, van Stekelenburg L, Pearson RB, Dziadek M, Smith PJ: STIM1: a novel phosphoprotein located at the cell surface. Biochim Biophys Acta 2000, 1481(1):147-155.
6. Lewis RS: The molecular choreography of a store-operated calcium channel. Nature 2007, 446(7133):284-287.
7. Wu MM, Buchanan J, Luik RM, Lewis RS: Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane. J Cell Biol 2006, 174(6):803-813.
8. Abdullaev IF, Bisaillon JM, Potier M, Gonzalez JC, Motiani RK, Trebak M: Stim1 and Orai1 mediate CRAC currents and store-operated calcium entry important for endothelial cell proliferation. Circ Res 2008, 103(11):1289-1299.
9. Park MS, Koff A: Overview of the cell cycle. Curr Protoc Cell Biol 2001, Chapter 8:Unit 8 1.
10. Firshein W: The DNA-membrane fraction of Pneumococcus contains a DNA replication complex. J Mol Biol 1972, 70(3):383-397.
11. Kastan MB, Bartek J: Cell-cycle checkpoints and cancer. Nature 2004, 432(7015):316-323.
12. Takuwa N, Zhou W, Kumada M, Takuwa Y: Ca(2+)-dependent stimulation of retinoblastoma gene product phosphorylation and p34cdc2 kinase activation in serum-stimulated human fibroblasts. J Biol Chem 1993, 268(1):138-145.
13. Flory MR, Moser MJ, Monnat RJ, Jr., Davis TN: Identification of a human centrosomal calmodulin-binding protein that shares homology with pericentrin. Proc Natl Acad Sci U S A 2000, 97(11):5919-5923.
14. Roderick HL, Cook SJ: Ca2+ signalling checkpoints in cancer: remodelling Ca2+ for cancer cell proliferation and survival. Nat Rev Cancer 2008, 8(5):361-375.
15. Gwack Y, Feske S, Srikanth S, Hogan PG, Rao A: Signalling to transcription: store-operated Ca2+ entry and NFAT activation in lymphocytes. Cell Calcium 2007, 42(2):145-156.
16. Buchholz M, Schatz A, Wagner M, Michl P, Linhart T, Adler G, Gress TM, Ellenrieder V: Overexpression of c-myc in pancreatic cancer caused by ectopic activation of NFATc1 and the Ca2+/calcineurin signaling pathway. In: EMBO J. vol. 25, 2006/07/29 edn; 2006: 3714-3724.
17. Downward J: Control of ras activation. Cancer Surv 1996, 27:87-100.
18. Woods D, Parry D, Cherwinski H, Bosch E, Lees E, McMahon M: Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1. Mol Cell Biol 1997, 17(9):5598-5611.
19. Marshall CJ: Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell 1995, 80(2):179-185.
20. Balmanno K, Cook SJ: Sustained MAP kinase activation is required for the expression of cyclin D1, p21Cip1 and a subset of AP-1 proteins in CCL39 cells. Oncogene 1999, 18(20):3085-3097.
21. Mossner E, Boll M, Pfleiderer G: Purification of human and bovine alkaline phosphatases by affinity chromatography. Hoppe Seylers Z Physiol Chem 1980, 361(4):543-549.
22. Wang J, Wang N, Xie J, Walton SC, McKown RL, Raab RW, Ma P, Beck SL, Coffman GL, Hussaini IM et al: Restricted epithelial proliferation by lacritin via PKCalpha-dependent NFAT and mTOR pathways. J Cell Biol 2006, 174(5):689-700.
23. Resjo S, Oknianska A, Zolnierowicz S, Manganiello V, Degerman E: Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response to serine/threonine phosphatase inhibitors: deactivation of PDE3B in vitro by protein phosphatase type 2A. Biochem J 1999, 341 ( Pt 3):839-845.
24. Ishihara H, Martin BL, Brautigan DL, Karaki H, Ozaki H, Kato Y, Fusetani N, Watabe S, Hashimoto K, Uemura D et al: Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. Biochem Biophys Res Commun 1989, 159(3):871-877.
25. Chuang JY, Wang YT, Yeh SH, Liu YW, Chang WC, Hung JJ: Phosphorylation by c-Jun NH2-terminal kinase 1 regulates the stability of transcription factor Sp1 during mitosis. Mol Biol Cell 2008, 19(3):1139-1151.
26. Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D, Goldberg AL: Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 1994, 78(5):761-771.
27. Craiu A, Gaczynska M, Akopian T, Gramm CF, Fenteany G, Goldberg AL, Rock KL: Lactacystin and clasto-lactacystin beta-lactone modify multiple proteasome beta-subunits and inhibit intracellular protein degradation and major histocompatibility complex class I antigen presentation. J Biol Chem 1997, 272(20):13437-13445.
28. Lee DH, Goldberg AL: Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol 1998, 8(10):397-403.
29. Attaix D, Combaret L, Pouch MN, Taillandier D: Regulation of proteolysis. Curr Opin Clin Nutr Metab Care 2001, 4(1):45-49.
30. Swingle M, Ni L, Honkanen RE: Small-molecule inhibitors of ser/thr protein phosphatases: specificity, use and common forms of abuse. Methods Mol Biol 2007, 365:23-38.
31. Hinds TD, Jr., Sanchez ER: Protein phosphatase 5. Int J Biochem Cell Biol 2008, 40(11):2358-2362.
32. Hanks SK, Ryzhova L, Shin NY, Brabek J: Focal adhesion kinase signaling activities and their implications in the control of cell survival and motility. Front Biosci 2003, 8:d982-996.
33. Playford MP, Schaller MD: The interplay between Src and integrins in normal and tumor biology. Oncogene 2004, 23(48):7928-7946.
34. Sontag JM, Sontag E: Regulation of cell adhesion by PP2A and SV40 small tumor antigen: an important link to cell transformation. Cell Mol Life Sci 2006, 63(24):2979-2991.
35. Jackson JL, Young MR: Protein phosphatase-2A modulates the serine and tyrosine phosphorylation of paxillin in Lewis lung carcinoma tumor variants. Clin Exp Metastasis 2002, 19(5):409-415.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2029-07-30起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2029-07-30起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw