(3.229.120.26) 您好!臺灣時間:2021/04/10 23:18
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:陳淑怡
研究生(外文):Chen Shu Yi
論文名稱:FocalAdhesionKinase在致癌基因Tpr-Met所引起細胞轉型過程中所扮演的角色
論文名稱(外文):Role of Focal Adhesion Kinase in Oncogene Tpr-Met-stimulated Cell Transformation
指導教授:陳鴻震
指導教授(外文):Chen Hong Chen
學位類別:碩士
校院名稱:國立中興大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
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:53
中文關鍵詞:FAKTpr-Met致癌基因磷酸化腫瘤細胞轉型
外文關鍵詞:FAKTpr-Metoncogenephosphorylationtumortransformation
相關次數:
  • 被引用被引用:0
  • 點閱點閱:71
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要
關鍵字:FAK、Tpr-Met、致癌基因、磷酸化、腫瘤、細胞轉型
Focal adhesion kinase(FAK)是一種細胞質內存在於focal contacts的酪氨酸磷酸化激酶,其分子量為125 kDa,目前被認為在integrin所調控的細胞功能中扮演一個關鍵性的角色。最近我們也發現,FAK的過量表現可與肝細胞生長因子的刺激產生協同作用,共同造成細胞的腫瘤化(Chan et al., 2002, J. Biol. Chem. 277, 50373-50379)。c-met是一種原致癌基因,其蛋白產物為肝細胞生長因子接受體,透過染色體轉位可產生具致癌性融合基因tpr-met。Tpr-Met是一種細胞質內分子量約為65 kDa的酪氨酸磷酸化激酶,其kinase是持續處於活化狀態,並且本身的酪氨酸呈現被磷酸化狀態。在本研究中,主要是探討Tpr-Met與FAK之間的作用。實驗結果顯示,細胞內Tpr-Met的表現會引起FAK酪氨酸磷酸化的上升,在in vitro Tpr-Met可以直接將FAK磷酸化。此外Tpr-Met可以直接地與FAK形成穩定的複合物,當Tpr-Met的Tyr-482和Tyr-489突變後則會減弱原本Tpr-Met與FAK的結合,並且也降低對FAK磷酸化的能力。我們也發現單獨FAK的N端或C端就可以與Tpr-Met形成穩定的複合物。更進一步探討FAK在Tpr-Met所引起細胞轉型上的影響,發現同時表現Tpr-Met和FAK蛋白會促進細胞anchorage-independent生長能力和侵犯能力,並且其侵犯能力與matrix metalloprotease-2活性和表現量的增加有關。生化的結果顯示FAK的存在對於Tpr-Met活化ERK、JNK、c-Jun 、AKT是重要的,但不增強原本Tpr-Met對STAT-3磷酸化的能力。綜合上述結果,FAK可能是Tpr-Met在增大下游訊號中的一個平臺,並且在Tpr-Met所引起的細胞轉型中扮演一個重要的角色。

Abstract
Keywords:FAK, Tpr-Met, oncogene, phosphorylation, tumor, transformation
Focal adhesion kinase (FAK), a 125 kDa cytoplasmic protein tyrosine kinase localized in focal contacts, plays a crucial role in the control of integrin- mediated cellular functions. We have previously demonstrated that increased expression of FAK renders epithelial cells susceptible to transformation by hepatocyte growth factor (HGF) stimulation (Chan et al., 2002, J. Biol. Chem. 277, 50373-50379). The HGF receptor is encoded by the proto-oncogene c-met, which can be uncongenially activated through a chromosomal rearrangement that creates a hybrid gene tpr-met. Tpr-Met, a 65 kDa protein tyrosine kinase which is constitutively active and phosphorylated on tyrosine residues. In this study, I attempted to examine the potential interaction between FAK and Tpr-Met. My results showed that Tpr-Met stimulates the tyrosine phosphorylation of FAK in intact cells and is capable of directly phosphorylating recombinant FAK in vitro. Moreover, I demonstrated that Tpr-Met is associated with FAK both in vivo and in vitro. Mutations at the Tyr-482 and Tyr-489 of Tpr-Met impair the ability of Tpr-Met to bind and phosphorylate FAK. The NH2- and COOH-terminal domains of FAK are sufficient for Tpr-Met binding in vivo and in vitro. My results showed that the expression of FAK enhances Tpr-Met induced anchorage-independent cell growth and cell invasiveness. The ability of these cells to invade Matrigel correlated with activation and increased expression of matrix metalloprotease-2. Biochemical analysis revealed that FAK is important for Tpr-Met-induced activation of ERK, JNK, c-Jun, and AKT, but not STAT-3. Together, FAK may serve as a platform for Tpr-Met to amplify the signals to the downstream and play an important role in Tpr-Met-induced cell transformation.

目錄
中文摘要…………………………………………………………………….……...1
英文摘要…………………………………………………………………….……...2
Ⅰ 前言…………………………………………………………………….……...3
Ⅱ 材料與方法……………………………………………………………………9
實驗材料
一、細胞株………………………………………………………………………….9
二、質體…………………………………………………………………..….…....10
三、抗體…………………………………………………………………..……….11
四、藥品…………………………………………………………………..……….12
五、儀器…………………………………………………………………………...14
實驗方法
一、細胞培養及細胞株的建立(Cell culture )……………………………….15
二、細胞蛋白質之萃取(Collection of cell lysate)……………….……….....16
三、免疫沈澱法(Immunoprecipitation)………………………………….......16
四、西方轉印法(Western blotting)……………………………..……....…....17
五、生物體外激酶活性試驗(In vitro kinase assay)………………….…......18
六、生物體外兩種蛋白的結合能力試驗(in vitro binding assay).................19
七、細胞軟洋菜膠生長分析(soft agar-colony formation assay)............….21
八、Matrigel侵犯分析(Matrigel invasion assay)…........................……....21
九、Matrix metalloprotease活性測試…………………………………………..22
Ⅲ 結果
一、Tpr-Met蛋白的表現會促進FAK蛋白上酪氨酸的磷酸化上升,並且可以
不受細胞貼附與否所調控。.....................................................................23
二、Tpr-Met所促進FAK酪氨酸的磷酸化上升,是可以不依賴Src激酶的活
性和FAK自我磷酸化的影響。…..............………....................................24
三、Tpr-Met磷酸化FAK上一些已知會被v-Src所磷酸化的酪氨酸的位置。.25
四、Tpr-Met上Tyrosine 482的存在對於能與FAK 結合形成穩定的複合物
是必要條件。………………...............……...........................……………..26
五、FAK的N或C端皆可以與Tpr-Met結合成複合物。…………….…….....27
六、FAK對於Tpr-Met引起細胞轉型的過程中扮演一個重要的角色。……….27
Ⅳ 討論………………………………………................……………............…..29
Ⅴ 參考文獻……………………………………………………………………….32
VI 圖表…………………………………………………………………………….39

REFERENCES
1. Cooper, C. S., Park, M., Blair, D. G., Tainsky, M. A., Huebner, K., Croce, C. M., and Vande Woude, G. F. 1984. Molecular cloning of a new transforming gene from a chemically transformed human cell line. Nature 311: 29-33.
2. Bottaro, D. P., Rubin, J. S., Faletto, D. L., Chan, A. M. L., Kmiecik, T. E., Vande Woude, G., and Aaronson, S. A. 1991. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 251: 802-804.
3. Nakopoulou, L., Gakiopoulou, H., and Keramopoulos, A. 2000. c-met tyrosine kinase receptor expression is associated with abnormal beta-catenin expression and favourable prognostic factors in invasive breast carcinoma. Histopathology 36: 313-325.
4. Umeki, K., Shiota, G., and Kawasaki, H. 1999. Clinical significance of c-met oncogene alterations in human colorectal cancer. Oncology 56: 314-321.
5. Kuniyasu, H., Yasui, W., and Kitadai, Y. 1992. Frequent amplification of the c-met gene in scirrhous type stomach cancer. Biochem Biophys Res Commun. 189: 227-232.
6. Kuniyasu, H., Yasui, W., and Yokozaki, H. 1993. Aberrant expression of c-met mRNA in human gastric carcinomas. Int. J. Cancer 55: 72-75.
7. Suzuki, K., Hayashi, N., and Yamada, Y. 1994. Expression of the c-met proto-oncogene in human hepatocellular carcinoma. Hepatology 20: 1231-1236.
8. Park, W. S., Dong, S. M., and Kim, S. Y. 1999. Somatic mutations in the kinase domain of the Met/hepatocyte growth factor receptor gene in childhood hepatocellular carcinomas. Cancer Res. 59: 307-310
9. Fischer, J., Palmedo, G., and von Knobloch, R. 1998. Duplication and over-expression of the mutant allele of the MET proto-oncogene in multiple hereditary papillary renal cell tumours. Oncogene 17:733-739.
10. Renzo, M. F., Narsimhan, R. P., and Olivero, M. 1991. Expression of the Met/HGF receptor in normal and neoplastic human tissues. Oncogene 6: 1997-2003.
11. Park, M., Dean, M., Cooper, C. S., Schmidt, M., O’Brien, J., Blair, D. G., and Woude, G. V. 1986. Mechanism of met oncogene activation. Cell 45: 895-904.
12. Chan, A. M. L., King, H. W. S., Tempest, P. R., Deakin, E. A., Cooper, C. S., and Brookes, P. 1987. Primary structure of the met protein tyrosine
kinase domain. Oncogene 1: 229-233.
13. Park, M., Dean, M., Kaul, K., Braun, M. J., Gonda, M. A., and Vande Woude, G. 1987. Sequence of met protooncogene cDNA has features characteristic of the tyrosine kinase family of growth-factor receptors. Proc. Natl. Acad. Sci. USA 84: 6379-6383.
14. Capetanaki, Y., Kuisk, I., Rothblum, K., and Starnes, S. 1990. Mouse vimentin: structural relationship to fos, jun, CREB, and tpr. Oncogene 5:645-655.
15. Landschulz, W. H., Johnson, P. F., and McKnight, S. L. 1988. The leucine zipper: a hypothetical structure common to a new class of DNA binding proteins. Science 240:1759-1764.
16. Rodrigues, G., and Park, M. 1993. Dimerization mediated through a Leucine zipper activates the oncogenic potential of the met receptor tyrosine kinase. Mol. Cell. Biol. 1: 6711-6722.
17. Lock, L. S., Frigault, M. M., Saucier, C., and Park, M. 2003. Grb2-independent recruitment of Gab1 requires the C-terminal lobe and structural integrity of the Met receptor kinase domain. J. Biol. Chem. 278: 30083-30090.
18. Furge, K. A., Zhang, Y. W., and Vande Woude, G. F. 2000. Met receptor tyrosine kinase: enhanced signaling through adapter proteins. Oncogene19: 5582—5589.
19. Soman, N. R., Correa, P., Ruiz, B., and Wogan, G. N. 1991. The tpr-met oncogenic rearrangement is present and expressed in human gastric carcinoma and precursor lesions. Proc. Natl. Acad. Sci. USA 88: 4892-4896.
20. Richardson, A., Malik, R. K., Hildebrand, J. D., and Parsons, J. T. 1997. Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: a role for paxillin tyrosine phosphorylation
Mol. Cell. Biol. 17: 6906-6914.
21. Richardson, A., and Parsons, J. T. 1996. A mechanism for regulation of the adhesion-associated proteintyrosine kinase pp125FAK. Nature 380: 538-540.
22. Cary, L. A., Chang, J. F., and Guan, J. L. 1996. Stimulation of cell migration by overexpression of focal adhesion kinase and its association with Src and Fyn. J. Cell Sci. 109: 1787-1794.
23. Ilic, D., Furuta, Y., Kanazawa, S., Takeda, N., Sobue, K., Nakatsuji, N., Nomura, S., Fujimoto, J., Okada, M., Yamamoto, T., and Aizawa, S. 1995. Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice. Nature 377: 539-544.
24. Oktay, M., Wary, K. K., Dans, M., Rirge, R. B., and Giancotti, F. G. 1999. Integrin-mediated activation of focal adhesion kinase is required for signaling to Jun NH2-terminal kinase and progression through the G1 phase of the cell cycle. J. Cell Biol. 145: 1461-1469.
25. Zhao, J. H., Reiske, H., and Guan, J. L. 1998. Regulation of the cell cycle by focal adhesion kinase. J. Cell Biol. 143:1997-2008.
26. Almeida, E. A. C., Ilic, D., Han, Q., Hauck, C. R., Jin, F., Kawakatsu, H., Schlaepfer, D. D., and Damsky, C. H. 2000. Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH2-terminal kinase. J. Cell Biol. 149: 741-754.
27. Chan, P. C., Lai, J. F., Cheng, C. H., Tang, M. J., Chiu, C. C., and Chen, H. C. 1999. Suppression of ultraviolet irradiation-induced apoptosis by overexpression of focal adhesion kinase in madin-darby canine kidney cells. J. Biol. Chem. 274: 26901-26906.
28. Frisch, S. M., Vuori, K., Ruoslahti, E., and Chan-Hui, P. Y. 1996. Control of adhesion-dependent cell survival by focal adhesion kinase. J. Cell Biol. 134: 793-799.
29. Ilic, D., Almeida, E. A. C., Schlaepfer, D. D., Dazin, P., Aizawa, S., and Damsky, C. H. 1998. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J. Cell Biol. 143: 547-560.
30. Cobb, B. S., Schaller, M. D., Leu, T. H., and Parsons, J. T. 1994. Stable association of pp60src and pp59fyn with the focal adhesion- associated protein tyrosine kinase, pp125FAK. Mol. Cell. Biol. 14:147-155.
31. Xing, Z., Chen, H. C., Nowlen, J. K., Taylor, S., 1994. Shalloway, D., and Guan, J. L. Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain. Mol. Biol. Cell 5: 413-421.
32. Chen, H. C., and Guan, J. L. 1994. Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase. Proc. Natl. Acad. Sci. USA 91: 10148-10152.
33. Chen, H. C., Appeddu, P. A., Isoda, H., and Guan, J. L. 1996. Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase. J. Biol. Chem. 271: 26329-26334.
34. Zhang, X., Chattopadhyay, A., Ji, Q. S., Owen, J. D., Ruest, P. J., Carpenter, G., and Hanks, S. T. 1999. Focal adhesion kinase promotes phospholipase C-γ1 activity. Proc. Natl. Acad. Sci. USA 96: 9021-9026.
35. Schlaepfer, D. D., Hanks, S. K., Hunter, T., and van der Geer, P. 1994. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature 372: 786-791.
36. Schlaepfer, D. D., and Hunter, T. 1996. Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases. Mol. Cell. Biol. 16: 5623-5633.
37. Polte, T. R., and Hanks, S. K. 1995. Interaction between focal adhesion kinase and Crk-associated tyrosine kinase substrate p130Cas. Proc. Natl. Acad. Sci. USA 92: 10678-10682.
38. Reiske, H. R., Kao, S. C., Cary, L. A., Guan, J. L., Lai, J. F., and Chen, H. C. 1999. Requirement of phosphatidylinositol 3-kinase in focal adhesion kinase-promoted aell migration. J. Biol. Chem. 274: 12361-12366.
39. Schlaepfer, D. D., and Hunter, T. 1997. Focal adhesion kinase overexpression enhances Ras-dependent integrin signaling to ERK2/Mitogen-activated protein kinase through Interactions with and Activation of c-Src. J. Biol. Chem. 272: 13189-13195.
40. Lai, J. F., Kao, S. C., Jiang, S. T., Tang, M. J., Chan, P. C., and Chen, H. C. 2000. Involvement of focal adhesion kinase in hepatocyte growth factor-induced scatter of madin-darby canine kidney cells. J. Biol. Chem. 275: 7474-7480.
41. Taylor, J. M., Hildebrand, J. D., Mack, C. P., Cox, M. E. and Parsons, J. T. 1998. Characterization of graf, the GTPase-activating protein for rho associated with focal adhesion kinase. Phosphorylation and possible regulation by mitogen-activated protein kinase. J. Biol. Chem. 273: 8063-8070.
42. Taylor, J. M., Macklem, M. M. and Parsons, J. T. 1999. Cytoskeletal changes induced by GRAF, the GTPase regulator associated with focal adhesion kinase, are mediated by Rho. J. Cell Sci. 112: 231-242.
43. Arold, S. T., Hoellerer, M. K. and Noble, M. E. 2002. The structural basis of localization and signaling by the focal adhesion targeting domain. Structure 10: 319-327.
44. Thomas, J. W., Cooley, M. A., Broome, J. M., Salgia, R., Griffin, J. D., Lombardo, C. R. and Schaller, M. D. 1999. The role of focal adhesion kinase binding in the regulation of tyrosine phosphorylation of paxillin. J.Biol. Chem. 274: 36684-36692.
45. Liu, S., Thomas, S. M., Woodside, D. G., Rose, D. M., Kiosses, W. B., Pfaff, M. and Ginsberg, M. H. 1999. Binding of paxillin to alpha4 integrins modifies integrin-dependent biological responses. Nature 402: 676-681.
46. Schaller, M. D., Otey, C. A., Hildebrand, J. D. and Parsons, J. T. 1995. Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains. J. Cell Biol. 130: 1181-1187.
47. Woodhouse, E. C., Chuaqui, R. F., and Liotta, L. A. 1997. General mechanisms of metastasis. American Cancer Society 80: 1529-1537.
48. Chan, P. C., Liang, C. C., Yu, K. C., Chang, M. C., Ho, W.L., Chen, B. H., and Chen, H. C. 2002. Synergistic effect of focal adhesion kinase overexpression and hepatocyte growth factor stimulation on cell transformation. J. Biol. Chem. 277:50373- 50379.
49. Paumelle R., Tulasne D., Kherrouche Z., Plaza S., Leroy C., Reveneau S., Vandenbunder B., Fafeur V., Tulashe D., and Reveneau S. 2002. Hepatocyte growth factor/scatter factor activates the ETS1 transcription factor by a RAS-RAF-MEKERK signaling pathway. Oncogene 21: 2309-2319.
50. Gerard A. R., Morag P., and Joseph S. 1997. Activation of the JNK pathway is essential for transformation by the Met oncogene. The EMBO Journal 16:2634-2645.
51. Xiao G. H., Jeffers M., Bellacosa A., Mitsuuchi Y., Vande Woude G. F., Testa J. R. 2001. Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways. Proc. Natl Acad. Sci. USA 98:247-252.
52. Boccaccio C., Ando M., Tamagnone L., Bardelli A., Michieli P., Battistini C., and Comoglio P. M. 1998. Induction of epithelial tubules by growth factor HGF depends on the STAT pathway. Nature 391:285-288.
53. Zhang, Y. W., Wang, L. M., Jove, R. and Vande Woude, G. F. 2002. Requirement of Stat3 signaling for HGF/SF-Met mediated tumorigenesis. Oncogene 21:217-226.
54. Frisch, S. M., Vuori, K., and Rouslahti, E. 1996. Control of adhesion- dependent cell survival by focal adhesion kinase. J. Cell Biol. 134: 793-799.
55. Weidner K. M., Di Cesare S., Sachs M., Brinkmann V., Behrens J., Birchmeier W. 1996. Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature. 1996 14:173-176.
56. Schaeper U., Gehring N. H., Fuchs K. P, Sachs M., Kempkes B., and Birchmeier W. 2000. Coupling of Gab1 to c-Met, Grb2, and Shp2 Mediates Biological Responses. J Cell Biol. 149:1419-32.
57. Lock L. S., Frigault M. M., Saucier C., and Park M. 2003. Grb2-independent recruitment of Gab1 requires the C-terminal lobe and structural integrity of the Met receptor kinase domain. J Biol. Chem. 278:30083-30090.

QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔