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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 周祖述(Tzuu-Shuh Jou) | |
dc.contributor.author | Jen-Yau Chen | en |
dc.contributor.author | 陳振耀 | zh_TW |
dc.date.accessioned | 2021-06-17T00:25:37Z | - |
dc.date.available | 2012-09-18 | |
dc.date.copyright | 2012-09-18 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-03-27 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/66206 | - |
dc.description.abstract | EBP50為一高度磷酸化的骨架蛋白(scaffold protein),其參與了許多細胞內的訊息傳導。磷酸化修飾與EBP50蛋白的功能調控有很密切的關係,已有數個蛋白磷酸酶(protein kinase)被證實具有磷酸化EBP50蛋白於特定絲胺酸(serine)定點的能力。本研究論文中,我們運用磷酸化專一性抗體(phospho-specific antibody)以及定點突變技術(site-directed mutagenesis),驗證了犬科動物EBP50蛋白上排序第347與348個絲胺酸可透過蛋白磷酸酶C(protein kinase C, PKC)修飾為磷酸化絲胺酸(phospho-serine)。隨著此二定點被磷酸化,EBP50蛋白在細胞內的分佈會從細胞膜上轉移至細胞質。同時,EBP50蛋白與ezrin蛋白以及β-PIX蛋白的結合關係亦會在此二絲胺酸被磷酸化後走向分離。目前已知,ezrin和β-PIX皆可扮演上游調控蛋白的角色,參與並促進Rac1蛋白的活化。當細胞大量表現突變蛋白以模擬EBP50蛋白於第347、348個絲胺酸被磷酸化時,該細胞內Rac1蛋白的活化會受到抑制。此外,當附著的細胞自培養皿基層剝離時,亦可觀察到此二絲胺酸的磷酸化修飾在懸浮的細胞中明顯地被誘發並增加。此現象被進一步釐清是蛋白磷酸酶C與蛋白去磷酸酶2A(protein phosphastase 2A,PP2A)兩者同時作用於此二絲胺酸並互相抵銷的最終結果。而懸浮細胞內Rac1蛋白的活化則會因為EBP50蛋白被磷酸化而受到抑制並導致細胞走向失巢凋亡(anoikis)。當細胞大量表現第347、348個絲胺酸無法被磷酸化的EBP50突變蛋白時,該細胞在懸浮狀態下則較能抵抗凋亡。綜觀本論文的研究結果,我們認為EBP50蛋白藉由磷酸化修飾的改變以及細胞內分布位置的轉換參與了Rac1蛋白的功能調控。 | zh_TW |
dc.description.abstract | EBP50 is a highly phosphorylated scaffold protein which participates in various cellular signalling pathways. Several protein kinases phosphorylate EBP50 at specific serine residues and the phosphorylation status of EBP50 is greatly related to its functional regulation. By site-directed mutagenesis and a phospho-specific antibody, we confirmed a protein kinase C (PKC)-dependent phosphorylation of EBP50 at canine serine 347 and 348. Increased phosphorylation at these two serine residues led to the relocation of EBP50 from plasma membrane to cytosol, and the dissociation of EBP50 from ezrin and β-PIX, which two upstream regulator of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to Rac1 activation. Further, detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation at serine 347 and 348, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation are remarkably resistant to apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T00:25:37Z (GMT). No. of bitstreams: 1 ntu-101-D95448004-1.pdf: 9132517 bytes, checksum: 8ebf2dce86c3e62ee83434a29d4a3e06 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | Table of Contents 1
Abstract 6 中文摘要 7 Abbreviations 8 Introduction 9 Materials and Methods 14 Plasmid construction 14 EBP50 knockdown by siRNA approach 14 Antibodies and reagents 15 Cell culture and transfection 16 Immunofluorescence and image acquisition 16 Immunoprecipitation 17 Western blotting analysis 17 Subcellular fractionation 18 Rac1 activation assay 19 GST pull-down assay 19 Cell scattering assay 19 In vitro dephosphorylation assay 20 Anoikis assay 20 Results 21 Phosphorylation of serine 347 and 348 induces the subcellular redistribution of EBP50 21 EBP50 is phosphorylated at both serine 347 and 348 in vivo 22 Phosphorylation of EBP50 at serine 347 and 348 modulates its interaction with ezrin 23 Serine 347 and 348 phosphorylation of EBP50 attenuates Rac1 activation in vivo 24 EBP50 phosphorylation at serine 347 and 348 modulates its interaction with β-PIX 27 Cell detachment from the substratum induces phosphorylation of EBP50 at serine 347/348 and down-regulates Rac1 activity by disassembly of the EBP50-β-PIX-ezrin complex 29 Discussion 33 Figures 38 Figure 1 Schematic diagram of EBP50 constructs 38 Figure 2 The cellular distribution of various EBP50 constructs 39 Figure 3 Amino acids alignment of ezrin-binding region in EBP50 of different species 40 Figure 4 Phosphorylation of serine 347 and 348 induces cytosolic relocation of EBP50 41 Figure 5 The reliability of EBP50 phospho-specific antibody (pEBP50(Ser347-8)) 42 Figure 6 PMA induces phosphorylation of EBP50 at both serine 347 and 348 in vivo 43 Figure 7 PMA induced cytosolic redistribution of EBP50 is accompanied by phosphorylation at serine 347 and 348 44 Figure 8 Phosphorylation of EBP50 serine 347 and 348 dissociates EBP50 from ezrin 45 Figure 9 The expression of EBP50 constructs has no effect on the apical distribution of ezrin 46 Figure 10 The involvement of EBP50 in Rac1 activation in vivo 47 Figure 11 siE50_16 cells overexpressing EBP50 mutant, mimicking serine 347/348 phosphorylation, become refractory to Rac1 activation 48 Figure 12 siE50_15 cells overexpressing EBP50 mutant, mimicking serine 347/348 phosphorylation, become refractory to Rac1 activation 49 Figure 13 Cells overexpressing EBP50 mutant, mimicking serine 347/348 phosphorylation are defective in cell scattering induced by MRC5 conditioned medium 50 Figure 14 The involvement of β-PIX in Rac1 activation induced by MRC5 conditioned medium 51 Figure 15 Dual phosphorylation of EBP50 serine 347/348 reduces EBP50 binding to β-PIX 52 Figure 16 Single phosphorylation of EBP50 at serine 347 or 348 barely affects EBP50 binding to β-PIX, and Rac1 activation induced by MRC5 conditioned medium 53 Figure 17 Rac1 activity of MDCK cells under attached and suspended conditions 54 Figure 18 Cell detachment from the substratum induces phosphorylation of EBP50 at serine 347/348, and the involvement of protein phosphatases in regulating EBP50 phosphorylation 55 Figure 19 Protein phosphatase 2A dephosphorylates EBP50 at serine 347/348 in vitro 56 Figure 20 The effects of EBP50 phosphorylation at serine 347/348 on Rac1 activity in suspended cells 57 Figure 21 The effects of EBP50 phosphorylation at serine 347/348 on anoikis by measuring sub-G1 population 58 Figure 22 The involvement of Rac1 in EBP50-regulated anoikis 59 Figure 23 Disassociation of the EBP50-ezrin-β-PIX complex in suspended cells 60 Figure 24 EBP50 phosphorylation affects anoikis of HaCaT cells 61 Figure 25 Schematic model depicting how loss of substratum interaction enhances phosphorylation of EBP50 at serine 347/348 and down-regulates Rac1 activity to induce anoikis 63 References 64 Appendix 71 | |
dc.language.iso | en | |
dc.title | EBP50磷酸化對Rac1活化與失巢凋亡之影響 | zh_TW |
dc.title | The Effects of EBP50 Phosphorylation on Rac1 Activation and Anoikis | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 呂勝春(Sheng-Chung Lee),李芳仁(Fang-Jen Lee),張智芬(Zee-Fen Chang),林蔚靖(Wey-Jinq Lin) | |
dc.subject.keyword | 失巢凋亡(anoikis),蛋白去磷酸酶,2A (PP2A),EBP50蛋白,Rac1蛋白,β-PIX蛋白, | zh_TW |
dc.subject.keyword | EBP50,Rac1,β-PIX,anoikis,PP2A, | en |
dc.relation.page | 82 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-03-27 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
顯示於系所單位: | 分子醫學研究所 |
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