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研究生:鄒文怡
研究生(外文):Wen-I Tsou
論文名稱:Rap1在T細胞活化上所扮演的角色
論文名稱(外文):The Role of the Small G Protein Rap1 in T cell Activation
指導教授:賴明宗賴明宗引用關係
指導教授(外文):Ming-Zong Lai
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:免疫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:68
中文關鍵詞:T細胞活化Rap1V12轉殖小鼠
外文關鍵詞:T cell activationRap1Rap1V12 transgenic mice
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Rap1會受到T細胞抗原受體 (TCR) 的活化,並在細胞中扮演調控黏著分子integrin的角色。然而Rap1在T細胞活化上所扮演的角色及影響黏著分子integrin時受到的調控機制,並無明確的結論。在本論文中,我們將分三個方面探討Rap1的角色。第一,探討CD28的刺激對於Rap1的活化。第二,利用表現抑制型Rap1N17及持續活化型Rap1V12的DO11.10細胞株,與建立Rap1V12轉殖基因小鼠來探討Rap1在T細胞活化時的影響。第三,了解p38 MAPK是否參與Rap1的訊息傳遞。
從我們的研究中發現,T細胞株EL4受到CD28的刺激會增加TCR對於Rap1的活化。而在表現Rap1V12的轉殖小鼠內,不影響胸腺T細胞與脾臟T細胞之發育(包含細胞數目、胸腺T細胞的正向選擇、CD4與CD8細胞之比例、TCR的表現)。而胸腺T細胞與脾臟T細胞受到活化後之細胞增生及IL-2的產量則較對照組高,顯示Rap1在T細胞內為正向調節分子。但在T 細胞株DO11.10內表現持續活化型Rap1V12及抑制型Rap1N17,其活化所產生的IL-2則未受到影響,也不影響DO11.10與抗原呈現細胞在有抗原存在下之結合比率。
在探討TCR活化Rap1的訊息傳遞上,我們發現T細胞株EL4以p38 MAPK特異抑制劑SB203580的預先處理後,會促進Rap1受到TCR刺激後的活性。但小鼠脾臟T細胞以SB203580的預先處理,增加了未活化T細胞內Rap1的活性,但經TCR及CD28刺激後則Rap1之活性並未明顯增加。因此,p38 MAPK調控Rap1的活化因不同細胞而異。
總結來說,我們的研究支持Rap1在正常細胞內扮演正向調節的角色。CD28的訊息會促進Rap1在T細胞內的活化。此外,p38 MAPK可能參與在TCR活化Rap1的訊息傳遞路徑中。我們同時也發現在不同的細胞中有相異的實驗結果,顯示調控Rap1的活化會因為在不同的細胞、細胞內的分布及活化量上的不同而有相當之複雜性。所以Rap1在T細胞活化及訊息傳導上所扮演的真正角色,需要更多研究來進一步釐清。
Rap1 is activated by TCR signaling and is participated in integrin activation. The exact role of Rap1 in T cell activation and the mechanism on how Rap1 regulates integrin activation have not been precisely defined. In this study, we explored the role of Rap1 in three different aspects. First, we examined the effect of CD28 co-stimulation on Rap1 activation. Second, we elucidated the role of Rap1 in T cell activation by expressing the dominant negative Rap1N17 and constitutive active Rap1V12 in DO11.10 hybridoma and Rap1V12 in transgenic mice. Third, we examined the possible involvement of p38 MAPK in Rap1 activation.
Results from our study illustrated CD28 co-stimulation increased Rap1 activation both in EL4 T lymphoma and splenic T cell, supporting a positive role of CD28. The T cell-specific transgenic expression of Rap1V12 did not affect T cell development, including cell number, CD4/CD8 ratio, positive selection and TCR levels. T cell proliferation and IL-2 production were increased in Rap1V12-transgenic mice, suggesting a positive role of Rap1 in T cell activation. In contrast, expression of Rap1V12 and Rap1N17 in DO11.10 T hybridoma did not affect CD3 or antigen peptide induced IL-2 production nor was antigen peptide-mediated T cell-antigen presenting cell conjugation.
In the study of the TCR signals that activate Rap1, we found that pretreatment with p38 MAPK specific inhibitor SB203580 increased TCR-stimulated Rap1 activation in EL4 cell. Pretreatment of splenic T cell with SB203580 resulted in increase Rap1 basal level not TCR-stimulated Rap1 activation. Therefore, the involvement of p38 MAPK in Rap1 activation is cell-type dependent.
In summary, our results clearly support a positive role of Rap1 in the activation of normal T cells. CD28 co-stimulation promoted the Rap1 activation. In addition, p38 MAPK may couple TCR activation to Rap1 activation. However, we also found exceptional results in different types of T cells, suggesting a complicated regulation of Rap1 which may depend on cell type, distribution, and quantity of Rap1. Further studies are required to delineate the exact role of Rap1 in T cell activation and signal coupling.
中文摘要 i
英文摘要 iii
縮寫對照表 viii

第一章 簡介
一、Rap1的發現 1
二、Rap1在細胞內之弁?
1.Rap1與Ras的交互作用 2
2.Rap1影響細胞型態與integrin的活化 4
3.Rap1與p38 MAPK的交互作用 6
研究目的 7

第二章 材料與方法
壹、細胞株與細胞培養
一、細胞株 8
二、小鼠胸腺與脾臟細胞 8
三、細胞培養 9
貳、藥品與試劑 9
參、抗體 9
肆、質體構築
一、pcDNA3-myc-Rap1 N17及pcDNA3-myc-Rap1 V12
質體 10
二、pGC-myc-Rap1 N17-YFP及pGC-myc-Rap1V12-YFP
質體 11
三、CD2-myc-Rap1 V12質體 11
伍、在DO11.10細胞表現Rap1突變株
一、以calcium phosphate轉染法將質體送入Phoenix細胞 11
二、以離心法感染DO11.10細胞 12
陸、基因轉殖鼠之建立
一、Genomic DNA測試 12
二、mRNA測試
1.以TRIzol試劑抽取細胞全RNA 13
2. RNA反轉錄反應 14
3. 反轉錄聚合�○s鎖反應 14
柒、T細胞增殖分析 14
捌、IL-2產量分析 15
玖、流式細胞儀分析
一、細胞表面染色分析 15
二、T細胞與B細胞結合分析
1.細胞染色 16
2.細胞結合 16
拾、純化GST 融合Ral-GDS RBD 之融合蛋白質 17
拾壹、Rap1活性之偵測 17
拾貳、西方墨點法 18

第三章 結果
一、Rap1在T細胞活化時的角色
1.CD28的訊息對於Rap1活化之影響 20
2.Rap1對於T細胞活化後IL-2分泌情形 21
3.Rap1對於T細胞與B細胞結合之影響 23
4.Rap1V12基因轉殖小鼠之分析 24
(1) Rap1V12基因轉殖小鼠T細胞和B細胞發育正常 25
(2) Rap1V12基因轉殖小鼠胸腺和脾臟T細胞活化後
,有較高程度的細胞增生及IL-2分泌 26
二、p38 MAP kinase在Rap1活化上的影響 27

第四章 討論
1. CD28對於Rap1活化之調控 30
2. Rap1對T細胞發育及活化之影響
(1) 在正常T細胞內Rap1對於其發育及活化影響 30
(2) 在DO11.10細胞株內Rap1對於其活化之影響 32
3. p38 MAPK對於TCR所造成Rap1活化之影響 33

圖表 35
參考文獻 60
Acuto O and Michel F. (2003) CD28-mediated co-stimulation: a quantitative support for TCR signaling. Nat Rev Immunol. 3:939-51
Agarwal S and Rao A. (1998) Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation. Immunity. 9:765-75
Amsen D, Kruisbeek A, Bos JL, Reedquist K. (2000) Activation of the Ras-related GTPase Rap1 by thymocyte TCR engagement and during selection. Eur J Immunol. 30:2832-41
Asha H, de Ruiter ND, Wang MG, Hariharan IK. (1999) The Rap1 GTPase functions as a regulator of morphogenesis in vivo. EMBO J. 18:605-15
Bivona TG, Wiener HH, Ahearn IM, Silletti J, Chiu VK, Philips MR. (2003) Rap up-regulation and activation on plasma membrane regulates T cell adhesion. J. Cell Biol. 164:461-70
Bos JL, de Rooij J, Reedquist KA. (2001) Rap1 signaling: adhering to new models. Nat Rev Mol Cell Biol. 2:369-77
Bos JL, de Bruyn K, Enserink J, Kuiperij B, Rangarajan S, Rehmann H, Riedl J, de Rooij J, van Mansfeld F, Zwartkruis F. (2003) The role of Rap1 in integrin-mediated cell adhesion. Biochem Soc Trans. 31:83-6
Boussiotis VA, Freeman GJ, Berezovskaya A, Barber DL, Nadler LM. (1997) Maintenance of human T cell anergy: blocking of IL-2 gene transcription by activated Rap1. Science. 278:124-8
Carey KD, Dillon TJ, Schmitt JM, Baird AM, Holdorf AD, Straus DB, Shaw AS, Stork PJ. (2000) CD28 and the Tyrosine Kinase Lck Stimulate Mitogen-Actived Protein Kinase Activity in T Cells via Inhibition of the Small G Protein Rap1. Mol Cell Biol. 20:8409-19
Caron, E. (2003) Cellular functions of the Rap1 GTP-binding protein: a patter emerges. J. cell Sci.116:435-40
Cook SJ, Rubinfeld B, Albert I, McCormick F. (1993) RapV12 antagonizes Ras-dependent activation of ERK1 and ERK2 by LPA and EGF in Rat-1 fibroblast. EMBO J. 12:3475-85
Czyzyk J, Leitenberg D, Taylor T, Bottomly K. (2000) Combinatorial effect of T-cell receptor ligation and CD45 isoform expression on the signaling contribution of the small GTPase Ras and Rap1. Mol Cell Biol. 20:8740-7
Delehanty LL, Mogass M, Gonias SL, Racke FK, Johnstone B, Goldfarb AN. (2003) Stromal inhibition of megakaryocytic differentiation is associated with blockade of sustained Rap1 activation. Blood. 101:1744-51
Dustin ML, Olszowy MW, Holdorf AD, Li J, Bromley S, Desai N, Widder P, Rosenberger F, van der Merwe PA, Allen PM, Shaw AS. (1998) A novel adaptor protein orchestrates receptor patterning and cytoskeletal polarity in T-cell contacts. Cell. 94:667-77
Dustin ML, Bivona TG, Philips MR. (2004) Membranes as messengers in T cell adhesion signaling. Nat Immunol. 5:363-72
Dong C, Davis RJ, Flavell RA. (2002) MAP kinases in the immune response. Annu Rev Immunol. 20:55-72
Franke B, Akkerman JW, Bos JL. (1997) Rapid Ca2+-mediated activation of Rap1 in human platelets. EMBO J.16:252-9
Grader-Beck T, van Puijenbroek AA, Nadler LM, Boussiotis VA. (2003) cAMP inhibits both Ras and Rap1 activation in primary T lymphocytes, but only Ras inhibition correlates with blockade of cell cycle progress. Blood. 101:998-1006
Hackeng CM, Franke B, Relou IA, Gorter G, Bos JL, van Rijn HJ, Akkerman JW. (2000) Low-density lipoprotein activates the small GTPases Rap1 and Ral in human platelets. Biochem J. 349:231-8
Hariharan IK, Carthew RW, Rubin GM. (1991) The Droshophila roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination. Cell. 67:717-22
Hattori M, Tsukamoto N, Nur-e-Kamal MS, Rubinfeld B, Iwai K, Kubota H, Maruta H, Minato N. (1995) Molecular cloning of a novel mitogen-inducible nuclear protein with a Ran GTPase-activating domain that affects cell cycle progression. Mol. Cell Biol. 15:552-60
Hattori M, Minato N. (2003) Rap1 GTPase: functions, regulation and malignancy. J Biochem (Tokyo) 134:479-84
Herrmann C, Horn G, Spaargaren M, Wittinghofer A. (1996) Differential interaction of the Ras family GTP-binding proteins H-Ras, Rap1A, and R-Ras with the putative effector molecules Raf kinase and Ral-guanine nucleotide exchange factor. J Biol Chem. 271:6794-800
Hynes, RO. (2002) Integrin: bidirectional, allosteric signaling machines. Cell. 110:673
Ishida D, Kometani K, Yang H, Kakugawa K, Masuda K, Iwai K, Suzuki M, Itohara S, Nakahata T, Hiai H, Kawamoto H, Hattori M, Minato N. (2003) Myeloproliferative stem cell disorders by deregulated Rap1 activation in SPA-1-deficeint mice. Cancer Cell. 4:55-65
Ishida D, Yang H, Masuda K, Uesugi K, Kawamoto H, Hattori M, Minato N. (2003) Antigen-driven T cell anergy and defective memory T cell response via deregulated Rap1 activation in SPA-1-defecient mice. Proc Natl Acad Sci U S A. 100:10919-24
Katagiri K, Hattori M, Minato N, Irie S, Takatsu K, Kinashi T. (2000) Rap1 is a potent activation signal for leukocyte function-associated antigen 1 distinct from protein kinase C and phophatidylinositol-3-OH kinase. Mol Cell Biol. 20:1956-69
Katagiri K, Hattori M, Minato N, Kinashi T. (2002) Rap1 functions as a key regulator of T-cell and antigen-presenting cell interactions and modulates T-cell responses. Mol Cell Biol. 22:1001-15
Katagiri K, Maeda A, Shimonaka M, Kinashi T. (2003) RapL, a Rap1-binding molecule that mediates Rap1-induced adhesion through spatial regulation of LFA-1. Nat Immunol. 4:741-8
Katagiri K, Shimonaka M, Kinashi T. (2004) Rap1-mediated lymphocyte function-associated antigen-1 activation by the T cell antigen receptor is dependent on phospholipase C-��1. J Biol Chem. 279:11875-81
Kawasaki H, Springett GM, Mochizuki N, Toki S, Nakaya M, Matsuda M, Housman DE, Graybiel AM. (1998) A family of cAMP-binding proteins that directly activate Rap1. Science. 282:2275-9
Kinbara K, Goldfinger LE, Hansen M, Chou FL, Ginsberg MH. (2003) Ras GTPase: integrins’ friends or foes? Nat Rev Mol Cell Biol. 4:767-76
Kitayama H, Sugimoto Y, Matsuzaki T, Ikawa Y, Noda M. (1989) A ras-related gene with transformation suppressor activity. Cell. 56:77-84
Mochizuki N, Ohba Y, Kiyokawa E, Kurata T, Murakami T, Ozaki T, Kitabatake A, Nagashima K, Matsuda M. (1999) Activation of the ERK/MAPK pathway by an isoform of rap1GAP associated with G��(i). Nature. 400:891-4
Mochizuki N, Yamashita S, Kurokawa K, Ohba Y, Nagai T, Miyawaki A, Matsuda M. (2001) Spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Nature. 411:1065-8
Okada T, Hu CD, Jin TG, Kariya K, Yamawaki-Kataoka Y, Kataoka T. (1999) The strength of interaction at the Raf cysteine –rich domain is a critical determinant of response of Raf to Ras family small GTPase. Mol Cell Biol. 19:6057-64
Palsson EM, Popoff M, Thelestam M, O''Neill LA. (2000) Divergent roles for Ras and Rap in the activation of p38 mitogen-activated protein kinase by interleukin-1. J Biol Chem. 275:7818-25
Park HO, Sanson A, Herskowitz I. (1999) Localization of bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site. Genes Dev. 13:1912-7
Pizon V, Chardin P, Lerosey I, Olofsson B, Tavitian A. (1988) Human cDNAs rap1 and rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the ‘effector’ region. Oncogene. 3:201-4
Rayter SI, Woodrow M, Lucas SC, Cantrell DA, Downward J. (1992) p21ras mediates control of IL-2 gene promoter function in T cell activation. EMBO J. 11:4549-56
Rebstein PJ, Cardelli J, Weeks G, Spiegelman GB. (1997) Mutational analysis of the role of Rap1 in regulating cytoskeletal function in Dictyostelium. Exp Cell Res. 231:276-83
Reedquist KA, Bos JL. (1998) Costimulation through CD28 suppresses T cell receptor-dependent activation of the Ras-like small GTPase Rap1 in human T lymphocytes. J Biol Chem. 273:4944-9
de Rooij J, Zwartkruis FJ, Verheijen MH, Cool RH, Nijman SM, Wittinghofer A, Bos JL. (1998) Epac is a Rap1 guanine-nucleotide-exchange factor directly activated by cyclic AMP. Nature. 396:474-7
Sawada Y, Nakamura K, Doi K, Takeda K, Tobiume K, Saitoh M, Morita K, Komuro I, De Vos K, Sheetz M, Ichijo H. (2001) Rap1 is involved in cell stretching modulation of p38 but not ERK or JNK MAP kinase. J Cell Sci. 114:1221-7
Schmidt A, Caron E, Hall A. (2001) Lipopolysaccharide-induced activation of ��2-integrin function in macrophages requires Irak kinase activity, p38 mitogen-activated protein kinase, and Rap1 GTPase. Mol Cell Biol. 21:438-48
Seastone DJ, Zhang L, Buczynski G, Rebstein P, Weeks G, Spiegelman G, Cardelli J. (1999) The small Mr Ras-like GTPase Rap1 and the phospholipase C pathway act to regulate phagocytosis in Dictyostelium discoideum. Mol Biol Cell. 10:393-406
Sebzda E, Bracke M, Tugal T, Hogg N, Cantrell DA. (2002) Rap1A positively regulates T cells via integrin activation rather than inhibiting lymphocyte signaling. Nat Immunol. 3:251-8
Shao Y, Elly C, Liu YC. (2003) Negative regulation of Rap1 activation by the Cbl E3 ubiquitin ligase. EMBO Rep. 4:425-31
Shimonaka M, Katagiri K, Nakayama T, Fujita N, Tsuruo T, Yoshie O, Kinashi T. (2003) Rap1 translates chemokine signals to integrin activation, cell polarization, and motility across vascular endothelium under flow. J Cell Biol. 161:417-27
Spaargaren M, Bischoff JR. (1994) Identification of the guanine nucleotide dissociation stimulator for Ral as a putative effector molecule of R-ras, H-ras, K-ras, and Rap. Proc Natl Acad Sci U S A. 91:12609-13
Stork PJ. (2003) Does Rap1 deserve a bad Rap? Trends Biochem Sci. 28:267-75
Swan KA, Alberola-Ila J, Gross JA, Appleby MW, Forbush KA, Thomas JF, Perlmutter RM. (1995) Involvement of p21ras distinguishes positive and negative selection in thymocytes. EMBO J. 14:276-85
Tohyama Y, Katagiri K, Pardi R, Lu C, Springer TA, Kinashi T. (2003) The critical cytoplasmic regions of the �埕/��2 integrin in Rap1-induced adhesion and migration. Mol Biol Cell. 14:2570-82
Tsukamoto N, Hattori M, Yang H, Bos JL, Minato N. (1999) Rap1 GTPase-activating protein SPA-1 negatively regulates cell adhesion. J Biol Chem. 274:18463-9
Vossler MR, Yao H, York RD, Pan MG, Rim CS, Stork PJ. (1997) cAMP activates MAP kinase and Elk-1 through a B-raf and Rap1-dependent pathway. Cell. 89:73-82
Yajnik V, Paulding C, Sordella R, McClatchey AI, Saito M, Wahrer DC, Reynolds P, Bell DW, Lake R, van den Heuvel S, Settleman J, Haber DA. (2003) DOCK4, a GTPase activator, is disrupted during tumorigenesis. Cell. 112:673-84
York RD, Yao H, Dillon T, Ellig CL, Eckert SP, McCleskey EW, Stork PJ. (1998) Rap1 mediates sustained MAP kinase activationo induced by nerve growth factor. Nature. 392:622-6
Zhu JJ, Qin Y, Zhao M, Van Aelst L, Malinow R. (2002) Ras and Rap control AMPA receptor trafficking during synaptic plasticity. Cell. 110: 443-55
Zwartkruis FJ, Wolthuis RM, Nabben NM, Franke B, Bos JL. (1998) Extracelluar signal-regulated activation of Rap1 fails to interfere in Ras effector signaling. EMBO J. 17:5905-12
Zwartkruis FJ, Bos JL. (1999) Ras and Rap1: two highly related small GTPase with distinct function. Exp Cell Res. 253:157-65
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