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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 賴明宗(Ming-Zong Lai) | |
dc.contributor.author | Pei-Yun Chang | en |
dc.contributor.author | 張沛昀 | zh_TW |
dc.date.accessioned | 2021-05-16T16:22:45Z | - |
dc.date.available | 2018-09-24 | |
dc.date.available | 2021-05-16T16:22:45Z | - |
dc.date.copyright | 2013-09-24 | |
dc.date.issued | 2013 | |
dc.date.submitted | 2013-07-21 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/6189 | - |
dc.description.abstract | DAPK是一個已知的抑癌蛋白。實驗室先前的研究發現,DAPK能在T細胞受體刺激之下活化,並且藉由抑制NF-κB路徑而阻礙T細胞活化。在更進一步的研究中指出,DAPK只會特定地抑制由T細胞受體所引發的NF-κB活化,並不影響TNFα或IL-1β所引發的NF-κB活化。DAPK抑制T細胞受體所引發的NF-κB活化是透過影響T細胞受體所引發的PKCθ磷酸化;然而,DAPK抑制PKCθ活化過程的機制目前仍然未被探討。在本研究中,我們探討DAPK如何抑制T細胞受體所引發的PKCθ磷酸化。最近的研究中指出,MAP4K3/GLK是在T細胞受體活化之下,負責磷酸化PKCθ的激酶。我們發現在HEK293T細胞及Jurkat T細胞之中,DAPK與PKCθ及MAP4K3/GLK皆具有交互作用。我們也發現DAPK與PKCθ及MAP4K3/GLK之間會以許多不同蛋白區位進行交互作用。在體外激酶實驗中,我們發現DAPK並不會磷酸化PKCθ及MAP4K3/GLK,但是DAPK會抑制MAP4K3/GLK活化所必需的自體磷酸化作用及其激酶活性。有研究中指出,T細胞受體所引發的MAP4K3/GLK活化過程必須經過SLP-76與MAP4K3/GLK進行交互作用才能達成。我們發現在HEK293T細胞及Jurkat T細胞之中,DAPK與SLP-76具有交互作用。在免疫沉澱實驗中,我們發現DAPK抑制SLP-76與MAP4K3/GLK在T細胞受體刺激之下所引發的交互作用。我們的結果顯示DAPK可能是利用不同的機制調控T細胞受體刺激所引發的PKCθ活化,一是透過降低MAP4K3/GLK激酶的活性,二是藉由抑制MAP4K3/GLK與上游鷹架蛋白SLP-76之間的連結而使MAP4K3/GLK無法活化,進而間接抑制其下游受質PKCθ的活化。DAPK如何抑制T細胞受體刺激所引發的NF-κB訊息,並專一性抑制PKCθ活化之詳細機制尚待更進一步的實驗探討。 | zh_TW |
dc.description.abstract | Death-associated protein kinase (DAPK) is well known as a tumor suppressor. Previous studies from our lab have shown that DAPK is activated after TCR stimulation. DAPK inhibits T cell activation through suppression of NF-κB signaling. Furthermore, DAPK specifically inhibits the TCR-induced NF-κB activation but not TNFα- or IL-1β-induced NF-κB activation. Results from our lab also demonstrated that DAPK inhibits TCR-induced PKCθ phosphorylation, but the exact biochemical process targeted by DAPK to inhibit PKCθ activation remains unclear. The specific aim of this study is to delineate the molecular mechanism on how DAPK inhibits TCR-induced PKCθ phosphorylation. MAP4K3/GLK is recently identified as the kinase that phosphorylates and activates PKCθ after TCR stimulation. We found that DAPK interacted with both PKCθ and MAP4K3/GLK in HEK293T cells and Jurkat JE6.1 T cells. We also identified several domains of DAPK that mediate the binding to PKCθ and MAP4K3/GLK. Using in vitro kinase assay, we found that DAPK did not phosphorylate PKCθ. DAPK did not phosphorylate MAP4K3/GLK, but DAPK inhibited MAP4K3/GLK auto-phosphorylation and kinase activity. We also found that DAPK bound SLP-76, the adaptor protein which binds MAP4K3/GLK and is required for MAP4K3/GLK activation. In vitro binding analysis demonstrated that the presence of DAPK decreased the binding between SLP-76 and MAP4K3/GLK. DAPK also inhibited the association between SLP-76 and MAP4K3/GLK in Jurkat JE6.1 T cells. Therefore, DAPK uses at least two different mechanisms to inhibit TCR-induced PKCθ activation: one by suppressing MAP4K3/GLK kinase activity, the other by reducing the binding of MAP4K3/GLK to SLP-76. Further characterization on the processes underlying the inactivation of MAP4K3/GLK and the reduced association of MAP4K3/GLK-SLP-76 by DAPK may help to understand the exact inhibitory mechanism of DAPK in TCR-induced NF-κB signaling. | en |
dc.description.provenance | Made available in DSpace on 2021-05-16T16:22:45Z (GMT). No. of bitstreams: 1 ntu-102-R00449011-1.pdf: 3194331 bytes, checksum: 22c23b41125d8954e88d8411afe564c2 (MD5) Previous issue date: 2013 | en |
dc.description.tableofcontents | 口試委員會審定書........................................................................................................ i
致謝............................................................................................................................... ii 摘要.............................................................................................................................. iv Abstract........................................................................................................................ v List of Abbreviations................................................................................................. vii Content......................................................................................................................... 1 Chapter I Introduction................................................................................................ 5 1.1 Death-associated protein kinase (DAPK)........................................................... 5 1.2 T cell activation.................................................................................................. 6 1.2.1 The signal transduction of T cell activation............................................. 6 1.2.2 Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76)................................................................................................... 8 1.2.3 Germinal center kinase-like kinase (MAP4K3/GLK).............................. 9 1.2.4 Protein kinase C θ (PKCθ)..................................................................... 10 1.2.5 DAPK inhibits T cell activation at the stage on or upstream of PKCθ... 11 1.3 Rationale........................................................................................................... 12 Chapter II Materials and Methods.......................................................................... 13 2.1 Cell lines and cell culture................................................................................. 13 2.1.1 Cell lines.................................................................................................13 2.1.2 Cell culture............................................................................................. 13 2.2 Antibodies......................................................................................................... 13 2.3 Chemicals and reagents.................................................................................... 14 2.4 Plasmid construction and preparation............................................................... 15 2.4.1 Construction of pcDNA4-hPKCθ-6xHA................................................15 2.4.2 Construction of pcDNA4-hMAP4K3/GLK-myc................................... 15 2.4.3 Construction of pcDNA4-hMAP4K3/GLK-6xHA................................ 16 2.4.4 Construction of truncated forms of human MAP4K3/GLK................... 16 2.4.5 Construction of pcDNA4-hSLP-76-6xHA............................................. 17 2.4.6 Construction of kinase-dead form of human PKCθ............................... 17 2.4.7 Construction of kinase-dead form of human MAP4K3/GLK................ 18 2.5 Transfection of plasmid DNA........................................................................... 18 2.5.1 Calcium phosphate transfection.............................................................18 2.5.2 Retrovirus infection................................................................................ 19 2.5.3 Electroporation....................................................................................... 19 2.6 T cell activation................................................................................................ 19 2.7 Western Blotting............................................................................................... 20 2.8 Immunoprecipitation........................................................................................ 20 2.9 Luciferase assay................................................................................................ 21 2.10 In vitro kinase assay........................................................................................ 22 2.11 In vitro binding assay...................................................................................... 22 Chapter III Results.................................................................................................... 23 3.1 Establishment of Jurkat JE6.1 T cell line stably expressing DAPK................. 23 3.2 DAPK inhibits TCR-stimulated NF-κB activation........................................... 23 3.2.1 Expression of DAPK mutant forms affects the activation of NF-κB reporter................................................................................................... 23 3.2.2 DAPK suppresses TCR-induced phosphorylation of PKCθ.................. 24 3.3 Interaction of DAPK with PKCθ...................................................................... 24 3.3.1 Immunoprecipitation analysis of DAPK and PKCθ............................... 24 3.3.2 Determination of the interaction domains between DAPK and PKCθ.. 25 3.4 Interaction of DAPK with GLK....................................................................... 25 3.4.1 Immunoprecipitation analysis of DAPK and GLK................................ 25 3.4.2 Determination of the interaction domains of DAPK and GLK.............. 26 3.5 To determine whether PKCθ or GLK is the substrate of DAPK...................... 27 3.5.1 Preparation of human PKCθ recombinant protein................................. 27 3.5.2 Preparation of human GLK recombinant protein................................... 27 3.5.3 Kinase assay performed with DAPK and PKCθ.................................... 27 3.5.4 Preparation of PKCθ and GLK kinase-dead recombinant proteins........ 28 3.5.5 DAPK does not phosphorylates PKCθ in vitro...................................... 28 3.5.6 DAPK does not phosphorylates GLK in vitro........................................ 29 3.6 To determine whether DAPK affect the kinase activity of GLK...................... 29 3.6.1 Kinase assay performed with GLK and MBP.........................................29 3.6.2 Preparation of human GLK recombinant protein................................... 30 3.6.3 Kinase assay performed with MAP4K3/GLK and SLP-76.................... 30 3.7 To determine whether DAPK interferes with the association of GLK and SLP-76............................................................................................................. 31 3.7.1 DAPK interacts with SLP-76................................................................. 32 3.7.2 The endogenous GLK-SLP-76 association is reduced by DAPK.......... 32 3.7.3 DAPK inhibits the in vitro binding between GLK and SLP-76..............32 Chapter IV Discussion.............................................................................................. 34 4.1 DAPK is associated with SLP-76, MAP4K3/GLK, and PKCθ........................ 34 4.2 DAPK kinase activity and inhibition of NF-κB activation............................... 35 4.3 DAPK inhibits the kinase activity of MAP4K3/GLK...................................... 35 4.4 DAPK inhibits the association of MAP4K3/GLK and SLP-76....................... 36 Chapter V Reference................................................................................................. 38 Chapter VI Figures................................................................................................. 40 Figure 1. Over-express DAPK in Jurkat JE6.1 T cell............................................. 40 Figure 2. DAPK inhibits TCR-stimulated NF-κB activation................................. 41 Figure 3. DAPK inhibits TCR-stimulated PKCθ phosphorylation........................ 42 Figure 4. Interaction of DAPK with PKCθ in HEK 293T cell............................... 43 Figure 5. Interaction of DAPK with PKCθ in Jurkat JE6.1 T cell......................... 44 Figure 6. The interaction between DAPK and PKCθ is mediated by multiple domains..................................................................................................... 45 Figure 7. The interaction between PKCθ and DAPK is mediated by multiple domains..................................................................................................... 46 Figure 8. Interaction of DAPK with GLK in HEK 293T cell................................ 47 Figure 9. Interaction of DAPK with GLK in Jurkat JE6.1 T cell........................... 48 Figure 10. The interaction between DAPK and GLK is mediated by multiple domains..................................................................................................... 49 Figure 11. The interaction between GLK and DAPK is mediated by multiple domains..................................................................................................... 50 Figure 12. Generation of the recombinant protein of PKCθ.................................. 51 Figure 13. Generation of the recombinant protein of GLK.................................... 52 Figure 14. Auto-phosphorylation of PKCθ............................................................. 53 Figure 15. Generation of the kinase-dead recombinant protein of GLK andPKCθ........................................................................................................ 54 Figure 16. PKCθ is not a substrate of DAPK......................................................... 55 Figure 17. GLK is not a substrate of DAPK........................................................... 56 Figure 18. The presence of DAPK reduces the kinase activity of GLK to MBP... 57 Figure 19. Generation of the recombinant protein of SLP-76................................ 58 Figure 20. The presence of DAPK reduces the kinase activity of GLK to SLP-76...................................................................................................... 59 Figure 21. Interaction of DAPK with SLP-76........................................................ 60 Figure 22. The association of GLK and SLP-76 is attenuated by DAPK in Jurkat JE6.1 T cells............................................................................................. 61 Figure 23. The association of GLK and SLP-76 is inhibited by DAPK in vitro.... 62 Figure 24. Model on DAPK-mediated inhibition of PKCθ activation................... 63 Chapter VII Appendix.............................................................................................. 64 Appendix 1. DAPK protein structure..................................................................... 64 Appendix 2. DAPK family..................................................................................... 65 Appendix 3. PKCθ protein structure...................................................................... 66 Appendix 4. GLK protein structure........................................................................ 67 Appendix 5. Signal transduction of TCR-induced T cell activation....................... 68 Appendix 6. NF-κB pathway.................................................................................. 69 Appendix 7. Diagram of GLK-induced PKCθ/NF-κB activation during TCR signaling................................................................................................... 70 Appendix 8. pET-21a map...................................................................................... 71 Appendix 9. pcDNA4/6xHA map..........................................................................72 Appendix 10. κB-Luc schematic diagram.............................................................. 73 | |
dc.language.iso | en | |
dc.title | DAPK抑制T細胞中PKCθ活化機轉之研究 | zh_TW |
dc.title | Study on how DAPK inhibits the activation of PKCθ in T cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 101-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 繆希椿(Shi-Chuen Miaw),李秀香(Hsiu-Hsiang Lee) | |
dc.subject.keyword | κ基因結合核因子,死亡相關蛋白激?蛋白酵素激?, | zh_TW |
dc.subject.keyword | NF-κB,DAPK,MAP4K3/GLK,PKCθ, | en |
dc.relation.page | 73 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2013-07-22 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
顯示於系所單位: | 免疫學研究所 |
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