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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 | 廖憶純(Yi-Chun Liao) | |
dc.contributor.author | Yen-Hui Pan | en |
dc.contributor.author | 潘妍卉 | zh_TW |
dc.date.accessioned | 2021-06-08T01:44:30Z | - |
dc.date.copyright | 2016-11-02 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-08-16 | |
dc.identifier.citation | Albasri A, Aleskandarany M, Benhasouna A, Powe DG, Ellis IO, Ilyas M, et al. (2011). CTEN (C-terminal tensin-like), a novel oncogene overexpressed in invasive breast carcinoma of poor prognosis. Breast Cancer Res. Tr. 126:47–54.
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Tensin3 is a negative regulator of cell migration and all four Tensin family members are downregulated in human kidney cancer. PLoS One 4: e4350 McBride KM, Banninger G, McDonald C, Reich NC. (2002). Regulated nuclear import of the STAT1 transcription factor by direct binding of importin-alpha. Embo. J. 21:1754-1763. McLean GW, Carragher NO, Avizienyte E, Evans J, Brunton VG, and Frame MC. (2005). The role of focal-adhesion kinase in cancer - a new therapeutic opportunity. Nature Reviews Cancer 5: 505-515 Nagano M, Hoshino D, Koshikawa N, Akizawa T, and Seiki M. (2012). Turnover of focal adhesions and cancer cell migration. International Journal of Cell Biology 2012: 310616 Nardozzi JD, Lott K, Cingolani G. (2010). Phosphorylation meets nuclear import: a review. Cell Communication and Signaling 8: 32 Sakashita K, Mimori K, Tanaka F, Kamohara Y, Inoue H, Sawada T, et al. (2008). Prognostic relevance of Tensin4 expression in human gastric cancer. Ann. Surg. Oncol.15: 2606–2613. 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Biochimica et Biophysica Acta 1692: 103-119 Wu TR, Hong YK, Wang XD, Ling MY, Dragoi AM, Chung AS, et al. (2002). SHP-2 is a dual-specificity phosphatase involved in Stat1 dephosphorylation at both tyrosine and serine residues in nuclei. J. Biol. Chem. 277:47572-47580. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19083 | - |
dc.description.abstract | C-terminal tensin-like (CTEN) 為集中附著點 (focal adhesion) 蛋白質 tensin 家族中的一員,它在細胞附著、遷移和癌症發生過程中扮演重要角色。CTEN 在大腸癌中有表現量上升的現象,並且會促進癌症的轉移及侵略等特性;此外,在腫瘤細胞的細胞核中也能偵測到高含量的 CTEN。本實驗室過去研究中證實了與非惡性細胞相比,CTEN 在癌細胞的細胞核中有明顯的累積。我們藉由比對 CTEN 蛋白質序列後發現,CTEN 並未帶有已知的 nuclear localization signal (NLS),而在第102~118 個胺基酸上則帶有 nuclear export signal (NES)。本研究藉由篩選出穩定表現 NES deletion (ΔNES) CTEN 的細胞株,分析其細胞形態、增生、貼附等能力,發現 CTEN 蛋白質序列上 NES 的缺失並未如預期的使 CTEN 在細胞核中有明顯的累積,而在大腸癌細胞株 SW480 細胞增生以及貼附能力上,與控制組相比亦無明顯差異。此外,本研究也探討 CTEN 磷酸化是否影響其於核質間穿梭,我們藉由質譜的方式發現 CTEN 的磷酸化位點位於 Thr347、Ser350 和 Ser386,而細胞核中的 CTEN 與細胞質中 CTEN 有相同的磷酸化位點,推測此磷酸化修飾可能不影響 CTEN 於細胞中的分佈情形。實驗進一步以 Kinasephos 2.0 探討 CTEN 上游可能的蛋白質激酶,並建構帶有磷酸化位點突變的 CTEN 質體,以探討 CTEN 磷酸化的調控機制與其對 CTEN 功能的影響。 | zh_TW |
dc.description.abstract | C-terminal tensin-like (CTEN) molecule, a member of the tensin family, is a focal adhesion protein that plays an important role in cell adhesion, cell migration and the development of tumorigenesis. Elevated CTEN level has been detected in colon cancer and that promotes the cancer metastasis and invasiveness. Furthermore, high CTEN expression is detected in the nucleus of cancer cell. We have confirmed that more CTEN accumulates in the nuclei of colon cancer cells than in non-malignant cells in our previous studies. We have also found that CTEN contains nuclear export signal (NES) between 102 to 118 amino acid residues by in silico analysis but no classical nuclear localization signal (NLS) on its sequence. In this study, by selection of cells stably expressing NES-deletion (ΔNES) CTEN, we found that CTEN does not remarkably accumulate in the nucleus when the NES was deleted and that the expression of ΔNES-CTEN does not significantly alter cell proliferation and cell adhesion in SW480 colon cancer cells. Moreover, we explore whether the phosphorylation of CTEN regulates the mechanisms underlying the subcellular distribution of CTEN. CTEN is phosphorylated on Thr347, Ser350, and Ser386. The mass spectrometry results showed that the phosphorylation residues identified on nuclear CTEN are the same as those on cytoplasmic CTEN, which suggested that phosphorylation of CTEN might not contribute to its localization. However, in silico analysis was carried out to identify kinases that might be responsible for phosphorylation of CTEN. We also constructed the CTEN plasmids with phosphorylation site mutations to elucidate the regulatory mechanisms underlying CTEN phosphorylation and its effects on CTEN’s function. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:44:30Z (GMT). No. of bitstreams: 1 ntu-105-R03b22003-1.pdf: 21515276 bytes, checksum: d6364630123f642cf7052a6f368c8c07 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | Abstract i
摘要 ii List of Abbreviations iii Table of Contents iv Chapter 1. Introduction 1 1.1 Focal Adhesion 1 1.2 Tensin Protein Family 1 1.3 The role of C-terminal Tensin-like (CTEN) Molecule in Cancers and its Nuclear Localization 2 1.4 Nuclear Localization Signal and Nuclear Export Signal 3 1.5 Phosphorylation in Nucleocytoplasmic Transport 5 1.6 Aims of This Study 6 Chapter 2. Materials and Methods 7 2.1 Cell Culture 7 2.1.1 Subculturing 7 2.1.2 Freezing and Thawing 8 2.1.3 Cell Counting 8 2.1.4 EGF Treatment 8 2.2 Plasmids and Transfection 9 2.2.1 CTEN Expression Plasmids 9 2.2.2 Polymerase Chain Reaction (PCR) 9 2.2.3 DNA Agarose Gel Electrophoresis 10 2.2.4 Transformation and Plasmid Purification 10 2.2.5 Transfection 11 2.3 Stable Cell Selection and Assays 11 2.3.1 Stable Cell Line Selection 11 2.3.2 Cell Adhesion Assay 12 2.3.3 Cell Proliferation Assay 12 2.3.4 IN Cell 2000 Analyzer 12 2.4 Subcellular Fractionation and Whole Cell Lysis 13 2.5 Protein Quantification 14 2.6 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 14 2.7 Western Blotting 15 2.7.1 Wet Transfer 15 2.7.2 Immunoblotting 15 2.7.3 Stripping 15 2.8 Immunoprecipitation (IP) 16 2.9 Phos-tagTM Acrylamide Gel Electrophoresis 16 2.10 Mass Spectrometry 17 2.10.1 In Gel Digestion 17 2.10.2 Phosphopeptide Enrichment 17 Chapter 3. Results 19 3.1 CTEN resides in the nucleus predominantly in colon cancer cells and contains nuclear export signal on its sequence 19 3.2 Stable expression of EGFP-fusion CTEN in SW480 colon cancer cells 20 3.3 CTEN is phosphorylated in colon cancer cells both in cytoplasmic and nuclear fractions 21 3.4 CTEN phosphorylation sites were further determined by mass spectrometry 22 3.5 Construction of phosphorylation sites mutation of CTEN and to discover possible kinases involved in CTEN phosphorylation 24 3.6 Phosphorylation sites mutation of CTEN does not alter CTEN’s subcellular localization and may not be involved in cell proliferation 25 Chapter 4. Discussions and Future Perspectives 27 References 31 Figures and Tables 35 | |
dc.language.iso | en | |
dc.title | CTEN 磷酸化與其於核質間穿梭之調控機制 | zh_TW |
dc.title | Regulatory mechanisms of phosphorylation and nucleocytoplasmic shuttling of CTEN | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張麗冠,謝淑貞,黃楓婷 | |
dc.subject.keyword | 核質分佈,磷酸化, | zh_TW |
dc.subject.keyword | CTEN,nucleocytoplasmic shuttling,phosphorylation, | en |
dc.relation.page | 72 | |
dc.identifier.doi | 10.6342/NTU201602361 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-08-16 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科技學系 | zh_TW |
顯示於系所單位: | 生化科技學系 |
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