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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 安形高志(Takashi Angata) | |
| dc.contributor.author | Chen Kah Jun | en |
| dc.contributor.author | 曾嘉建 | zh_TW |
| dc.date.accessioned | 2022-11-23T09:08:12Z | - |
| dc.date.available | 2026-08-25 | |
| dc.date.available | 2022-11-23T09:08:12Z | - |
| dc.date.copyright | 2021-09-11 | |
| dc.date.issued | 2021 | |
| dc.date.submitted | 2021-08-25 | |
| dc.identifier.citation | 1. Tonks, N.K. Protein tyrosine phosphatases: from genes, to function, to disease. Nature Reviews Molecular Cell Biology 7, 833-846 (2006). 2. Denu, J.M. Dixon, J.E. Protein tyrosine phosphatases: mechanisms of catalysis and regulation. Curr Opin Chem Biol 2, 633-641 (1998). 3. Hunter, T. Protein kinases and phosphatases: The Yin and Yang of protein phosphorylation and signaling. Cell 80, 225-236 (1995). 4. Guan, K.L., Broyles, S.S. Dixon, J.E. A Tyr/Ser protein phosphatase encoded by vaccinia virus. Nature 350, 359-362 (1991). 5. Fauman, E.B. Saper, M.A. Structure and function of theprotein tyrosine phosphatases. Trends in Biochemical Sciences 21, 413-417 (1996). 6. Wang, Y. Pallen, C.J. The receptor-like protein tyrosine phosphatase HPTP alpha has two active catalytic domains with distinct substrate specificities. Embo j 10, 3231-3237 (1991). 7. Wu, L., Buist, A., den Hertog, J. Zhang, Z.-Y. Comparative Kinetic Analysis and Substrate Specificity of the Tandem Catalytic Domains of the Receptor-like Protein-tyrosine Phosphatase α*. Journal of Biological Chemistry 272, 6994-7002 (1997). 8. Bilwes, A.M., den Hertog, J., Hunter, T. Noel, J.P. Structural basis for inhibition of receptor protein-tyrosine phosphatase-α by dimerization. Nature 382, 555-559 (1996). 9. Zheng, X.M., Wang, Y. Pallen, C.J. Cell transformation and activation of pp60c-src by overexpression of a protein tyrosine phosphatase. Nature 359, 336-339 (1992). 10. Volberg, T., et al. The effect of tyrosine-specific protein phosphorylation on the assembly of adherens-type junctions. Embo j 11, 1733-1742 (1992). 11. Matsuyoshi, N., et al. Cadherin-mediated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts. J Cell Biol 118, 703-714 (1992). 12. Roura, S., Miravet, S., Piedra, J., García de Herreros, A. Duñach, M. Regulation of E-cadherin/Catenin association by tyrosine phosphorylation. J Biol Chem 274, 36734-36740 (1999). 13. Xie, Y., et al. Quantitative profiling of spreading-coupled protein tyrosine phosphorylation in migratory cells. Sci Rep 6, 31811 (2016). 14. Parsons, J.T. Focal adhesion kinase: the first ten years. J Cell Sci 116, 1409-1416 (2003). 15. Schlaepfer, D.D., Mitra, S.K. Ilic, D. Control of motile and invasive cell phenotypes by focal adhesion kinase. Biochim Biophys Acta 1692, 77-102 (2004). 16. Cohen, L.A. Guan, J.L. Mechanisms of focal adhesion kinase regulation. Curr Cancer Drug Targets 5, 629-643 (2005). 17. Young, K.A., Biggins, L. Sharpe, H.J. Protein tyrosine phosphatases in cell adhesion. Biochemical Journal 478, 1061-1083 (2021). 18. Yao, Z., et al. A Global Analysis of the Receptor Tyrosine Kinase-Protein Phosphatase Interactome. Molecular Cell 65, 347-360 (2017). 19. Su, J., Muranjan, M. Sap, J. Receptor protein tyrosine phosphatase α activates Src-family kinases and controls integrin-mediated responses in fibroblasts. Current Biology 9, 505-511 (1999). 20. Chen, M., Chen, S.C. Pallen, C.J. Integrin-induced Tyrosine Phosphorylation of Protein-tyrosine Phosphatase-α Is Required for Cytoskeletal Reorganization and Cell Migration*. Journal of Biological Chemistry 281, 11972-11980 (2006). 21. Sun, G., Cheng, S.Y.S., Chen, M., Lim, C.J. Pallen, C.J. Protein Tyrosine Phosphatase #x3b1; Phosphotyrosyl-789 Binds BCAR3 To Position Cas for Activation at Integrin-Mediated Focal Adhesions. Molecular and Cellular Biology 32, 3776-3789 (2012). 22. Zheng, X.-M., Resnick, R.J. Shalloway, D. A phosphotyrosine displacement mechanism for activation of Src by PTPα. The EMBO Journal 19, 964-978 (2000). 23. den Hertog, J., Tracy, S. Hunter, T. Phosphorylation of receptor protein-tyrosine phosphatase alpha on Tyr789, a binding site for the SH3-SH2-SH3 adaptor protein GRB-2 in vivo. The EMBO Journal 13, 3020-3032 (1994). 24. Cheng, S.Y.S., Sun, G., Schlaepfer, D.D. Pallen, C.J. Grb2 Promotes Integrin-Induced Focal Adhesion Kinase (FAK) Autophosphorylation and Directs the Phosphorylation of Protein Tyrosine Phosphatase #x3b1; by the Src-FAK Kinase Complex. Molecular and Cellular Biology 34, 348-361 (2014). 25. Hunter, T. A thousand and one protein kinases. Cell 50, 823-829 (1987). 26. Mustelin, T. Taskén, K. Positive and negative regulation of T-cell activation through kinases and phosphatases. Biochem J 371, 15-27 (2003). 27. den Hertog, J. Hunter, T. Tight association of GRB2 with receptor protein-tyrosine phosphatase alpha is mediated by the SH2 and C-terminal SH3 domains. Embo j 15, 3016-3027 (1996). 28. den Hertog, J., Tracy, S. Hunter, T. Phosphorylation of receptor protein-tyrosine phosphatase alpha on Tyr789, a binding site for the SH3-SH2-SH3 adaptor protein GRB-2 in vivo. Embo j 13, 3020-3032 (1994). 29. Su, J., Batzer, A. Sap, J. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2. J Biol Chem 269, 18731-18734 (1994). 30. Su, J., Yang, L.T. Sap, J. Association between receptor protein-tyrosine phosphatase RPTPalpha and the Grb2 adaptor. Dual Src homology (SH) 2/SH3 domain requirement and functional consequences. J Biol Chem 271, 28086-28096 (1996). 31. M⊘ller, N.P.H., et al. Selective Down-regulation of the Insulin Receptor Signal by Protein-tyrosine Phosphatases α and ∊(∗). Journal of Biological Chemistry 270, 23126-23131 (1995). 32. Kapp, K., Metzinger, E., Kellerer, M., Häring, H.-U. Lammers, R. The protein tyrosine phosphatase α modifies insulin secretion in INS-1E cells. Biochemical and Biophysical Research Communications 311, 361-364 (2003). 33. Buist, A., Blanchetot, C., Tertoolen, L.G.J. den Hertog, J. Identification of p130 cas as an in Vivo Substrate of Receptor Protein-tyrosine Phosphatase; *. Journal of Biological Chemistry 275, 20754-20761 (2000). 34. den Hertog, J., et al. Receptor protein tyrosine phosphatase alpha activates pp60c-src and is involved in neuronal differentiation. Embo j 12, 3789-3798 (1993). 35. Su, J., Muranjan, M. Sap, J. Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts. Curr Biol 9, 505-511 (1999). 36. Ponniah, S., Wang, D.Z.M., Lim, K.L. Pallen, C.J. Targeted disruption of the tyrosine phosphatase PTPα leads to constitutive downregulation of the kinases Src and Fyn. Current Biology 9, 535-S531 (1999). 37. Navada, S., Lai, P., Schwartz, A.G. Kalemkerian, G.P. Temporal trends in small cell lung cancer: Analysis of the national Surveillance, Epidemiology, and End-Results (SEER) database. Journal of Clinical Oncology 24, 7082-7082 (2006). 38. Sher, T., Dy, G.K. Adjei, A.A. Small cell lung cancer. Mayo Clin Proc 83, 355-367 (2008). 39. Shaper, A.G., Wannamethee, S.G. Walker, M. Pipe and cigar smoking and major cardiovascular events, cancer incidence and all-cause mortality in middle-aged British men. Int J Epidemiol 32, 802-808 (2003). 40. Henley, S.J., Thun, M.J., Chao, A. Calle, E.E. Association between exclusive pipe smoking and mortality from cancer and other diseases. J Natl Cancer Inst 96, 853-861 (2004). 41. Boffetta, P. Human cancer from environmental pollutants: the epidemiological evidence. Mutat Res 608, 157-162 (2006). 42. Hackshaw, A.K., Law, M.R. Wald, N.J. The accumulated evidence on lung cancer and environmental tobacco smoke. Bmj 315, 980-988 (1997). 43. Freudenheim, J.L., et al. Alcohol consumption and risk of lung cancer: a pooled analysis of cohort studies. Am J Clin Nutr 82, 657-667 (2005). 44. Künzli, N. Tager, I.B. Air pollution: from lung to heart. Swiss Med Wkly 135, 697-702 (2005). 45. Boffetta, P. Epidemiology of environmental and occupational cancer. Oncogene 23, 6392-6403 (2004). 46. Hwang, S.J., et al. Lung cancer risk in germline p53 mutation carriers: association between an inherited cancer predisposition, cigarette smoking, and cancer risk. Hum Genet 113, 238-243 (2003). 47. Li, X. Hemminki, K. Inherited predisposition to early onset lung cancer according to histological type. Int J Cancer 112, 451-457 (2004). 48. Bailey-Wilson, J.E., et al. A major lung cancer susceptibility locus maps to chromosome 6q23-25. Am J Hum Genet 75, 460-474 (2004). 49. Thorgeirsson, T.E., et al. A variant associated with nicotine dependence, lung cancer and peripheral arterial disease. Nature 452, 638-642 (2008). 50. Hung, R.J., et al. A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25. Nature 452, 633-637 (2008). 51. Molina, J.R., Yang, P., Cassivi, S.D., Schild, S.E. Adjei, A.A. Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 83, 584-594 (2008). 52. Cox, J. Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat Biotechnol 26, 1367-1372 (2008). 53. Tyanova, S., Temu, T. Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat Protoc 11, 2301-2319 (2016). 54. Hornbeck, P.V., et al. PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res 40, D261-270 (2012). 55. Hornbeck, P.V., et al. 15 years of PhosphoSitePlus®: integrating post-translationally modified sites, disease variants and isoforms. Nucleic Acids Res 47, D433-d441 (2019). 56. Zaidel-Bar, R., Itzkovitz, S., Ma'ayan, A., Iyengar, R. Geiger, B. Functional atlas of the integrin adhesome. Nat Cell Biol 9, 858-867 (2007). 57. Dingemans, A.-M.C., et al. Integrin expression profiling identifies integrin alpha5 and beta1 as prognostic factors in early stage non-small cell lung cancer. Molecular Cancer 9, 152 (2010). 58. Hornbeck, P.V., et al. PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic acids research 40, D261-D270 (2012). 59. Zaidel-Bar, R., Itzkovitz, S., Ma'ayan, A., Iyengar, R. Geiger, B. Functional atlas of the integrin adhesome. Nature cell biology 9, 858-867 (2007). 60. Valgeirsdottir, S., et al. PDGF induces reorganization of vimentin filaments. Journal of Cell Science 111, 1973-1980 (1998). 61. Horton, E.R., et al. Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly. Nat Cell Biol 17, 1577-1587 (2015). 62. Zeng, L., et al. PTP alpha regulates integrin-stimulated FAK autophosphorylation and cytoskeletal rearrangement in cell spreading and migration. J Cell Biol 160, 137-146 (2003). 63. Stanford, S.M., et al. Receptor Protein Tyrosine Phosphatase α-Mediated Enhancement of Rheumatoid Synovial Fibroblast Signaling and Promotion of Arthritis in Mice. Arthritis Rheumatol 68, 359-369 (2016). 64. Yang, C.Y., et al. Src and SHP2 coordinately regulate the dynamics and organization of vimentin filaments during cell migration. Oncogene 38, 4075-4094 (2019). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79705 | - |
| dc.description.abstract | 在此研究中，我試圖通過將蛋白酪胺酸磷酸酶受體型A（PTPRA）敲除和對照組細胞的比較來分析鑒定非小型肺癌細胞（NCI- H1975）中的PTPRA底物。當非小型肺癌細胞的PTPRA蛋白被敲除後，我發現細胞的貼附性跟對照組相較起來變得比較差，因此這個原因驅使我從細胞黏附的方向下去尋找PTPRA之底物。首先，我利用西方式點墨法和流式細胞術來比較對照組及PTPRA KO組細胞之上游整合素（Integrins）的表現量著手。透過實驗，我們發現對照組及PTPRA KO組細胞之間的整合素表現量幾乎沒有區別。接下來，我選用了曾經有被文獻報導過跟細胞貼附相關的PTPRA底物之蛋白來比較對照組和PTPRA KO組細胞的蛋白質酪胺酸磷酸化之間的差異。結果，我發現PTPRA KO細胞的FAK及P130Cas蛋白磷酸化表現量與對照細胞相較之下比較高，這個狀況意味著這些蛋白可能為PTPRA下游之效應物。緊接著，我利用酪胺酸磷酸化蛋白的免疫沈澱法及蛋白質組學分析法去鑒定PTPRA之底物。我觀察到PTPRA KO細胞在60 kDa左右大小的位置相較於對照組表現出比較強烈的訊號，所以我們決定探討這個位置的未知蛋白。透過質譜分析，我們成功找到了數個跟細胞貼附相關，而且可能在PTPRA KO細胞中酪胺酸磷酸化較為強烈的蛋白。綜觀以上結果，本研究揭示了數個可能為PTPRA底物的蛋白或許可以解釋PTPRA KO的表型，同時未來也值得去做更深入的探討。 | zh_TW |
| dc.description.provenance | Made available in DSpace on 2022-11-23T09:08:12Z (GMT). No. of bitstreams: 1 U0001-2408202111051400.pdf: 8122303 bytes, checksum: a4893271bbf97c11cb1abf6ef1b43dcb (MD5) Previous issue date: 2021 | en |
| dc.description.tableofcontents | "ACKNOWLEDGEMENTS I 中文摘要 II ABSTRACT III INTRODUCTION 1 Protein Tyrosine Phosphatase (PTP) 1 Protein Tyrosine Phosphatase Receptor-type A (PTPRA) 2 Focal Adhesion 3 Signalling Transduction 4 Possible substrates for PTPRA 5 Non-Small Cell Lung Cancer (NSCLC) 5 Preliminary Work in our lab 7 SPECIFIC AIM 10 MATERIALS AND METHODS 11 Chemical compounds and cell lines 11 Cell subculture 11 Cell Count 12 Protein Sample Preparation #1 12 Protein Sample Preparation #2 (with fibronectin stimulation of the cells) 13 Fibronectin-coating plate 13 Flow cytometry 14 Lentivirus Transduction 14 Immunoprecipitation of tyrosine-phosphorylated proteins 15 Silver Staining 16 In-gel digestion 16 Mass Spectrometry Analysis 18 Microscopy 20 Antibodies 20 Immunoblotting analysis 21 RESULTS 23 Lentivirus transduction to prepare H1975 sgRNA control cells and a new batch of PTPRA KO cells. 23 H1975 lung cancer PTPRA KO cells are less adherent to culture dish compared to sgRNA control cells. 23 Evaluation of the expression levels of integrins on the newly prepared H1975 sgRNA control cells and PTPRA KO cells by western blotting. 24 Evaluation of the expression levels of integrins on sgRNA control cells and PTPRA KO cells by flow cytometry. 25 Focal adhesion signalling is upregulated in PTPRA KO cells compared to sgRNA control cells. 26 A stronger band around 60 kDa was detected by anti-phosphotyrosine antibody in PTPRA KO cells. 27 Immunoprecipitation and silver staining for sgRNA control and PTPRA KO cells. 27 Mass spectrometry analysis and informatic screening of possible substrates for PTPRA. 28 DISCUSSIONS 29 Summary of results 29 Altered expression and tyrosine phosphorylation of FAK and p130Cas in PTPRA KO cells. 29 Verification of PTPRA substrate(s) identified by mass spectrometry is required. 30 H1975 lung cancer cells that overexpress PTPRA may be useful as a complementary sample. 31 Verification of vimentin as a tyrosine-phosphorylated protein downstream of PTPRA. 31 Validate that glycosylation of PTPRA may influence its function. 32 Possible mechanistic explanation of the relation between PTPRA and vimentin. 33 Technical Issue that needs to be overcome in the future. 34 FIGURES AND TABLES 35 Figure 1. Flow chart of lentivirus transduction for H1975 lung cancer cells. 36 Figure 2. Cells adhesion was observed using light microscopy. 38 Figure 3. The expression of various integrins in PTPRA KO and sgRNA control H1975 cells as revealed by western blotting. 40 Figure 4. The expression of various integrins on the surface of PTPRA KO and sgRNA control H1975 cells as detected by flow cytometry. 43 Figure 5. Expression and tyrosine phosphorylation of PTPRA substrate candidates in sgRNA control and PTPRA KO cells detected by immunoblotting analysis. 45 Figure 6. Comparing tyrosine phosphorylation with pY20, pY100, and 4G10 antibodies between sgRNA control cells and PTPRA KO cells at different time points by western blot analysis. 47 Figure 7. Silver staining of the proteins immunoprecipitated with 4G10 antibody from sgRNA control and PTPRA KO cell lysates. 48 Figure 8. Mass Spectrometry Analysis of PTPRA substrates. 49 SUPPLEMENTARY DATA 50 REFERENCES 56 " | |
| dc.language.iso | en | |
| dc.subject | 黏著信號傳遞 | zh_TW |
| dc.subject | 非小型肺癌細胞 | zh_TW |
| dc.subject | 磷酸酶PTPRA | zh_TW |
| dc.title | 探討參與非小型肺癌細胞黏著信號傳遞的磷酸酶PTPRA之受體 | zh_TW |
| dc.title | Identifying PTPRA substrates involved in focal adhesion signalling of non-small cell lung cancer | en |
| dc.date.schoolyear | 109-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 孟子青(Hsin-Tsai Liu),邱繼輝(Chih-Yang Tseng) | |
| dc.subject.keyword | 磷酸酶PTPRA,黏著信號傳遞,非小型肺癌細胞, | zh_TW |
| dc.subject.keyword | PTPRA,Focal Adhesion Signalling,Non-small cell lung cancer, | en |
| dc.relation.page | 63 | |
| dc.identifier.doi | 10.6342/NTU202102662 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2021-08-25 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| dc.date.embargo-lift | 2026-08-25 | - |
| 顯示於系所單位： | 生化科學研究所 | |
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