用FRET方法研究与Mps1蛋白有相互作用的CENP-E蛋白结构域

中国生物工程杂志

2009, Vol. 29

Issue (04): 28-34

研究报告

用FRET方法研究与Mps1蛋白有相互作用的CENP-E蛋白结构域

刘子杰,翁亚光,李素彦,施琼,蔡燕,刘斌,张燕,阎琛

重庆医科大学医学检验系临床检验诊断学省部共建教育部重点实验室重庆市重点实验室重庆

Explore the Structural Domains Of CENP-E Protein Interacting with Mps1 protein by FRET method

全文: PDF(803 KB) HTML

摘要： 目的：探索与Mps1蛋白有相互作用的CENP-E蛋白结构域。方法：将重组质粒pEGFP-CENPE2（674~1085位氨基酸）、pEGFP-CENPE3（1200~2134位氨基酸）转染人胚肾293(HEK293)细胞,采用受体漂白荧光共振能量转移方法（FRET方法）,检测EGFP-CENPE2、EGFP-CENPE3和Mps1间的能量转移率(Ef), 进一步用免疫共沉淀方法验证FRET的实验结果。结果：重组质粒转染HEK293细胞后经激光共聚焦显微镜观察重组质粒表达的融合蛋白与Mps1都存在着共定位；FRET检测结果显示EGFP-CENPE3和Mps1间的能量转移率为［(12.63±0.48)%, n=30］,pEGFP-CENPE2和Mps1间的能量转移率为［(3.07±0.21)%, n=30］，与对照组［(2.96±0.27)%, n=30］比较pEGFP-CENPE3和Mps1间的能量转移率差异存在显著性（p<0.05），免疫共沉淀实验结果显示EGFP-CENPE3与Mps1蛋白间存在相互作用。结论：FRET技术和免疫共沉淀实验证明了EGFP-CENPE3与Mps1间存在着相互作用。

关键词： CENP-E;Mps1;蛋白质相互作用;荧光共振能量转移

Abstract: Objective: To explore the structural domains of the CENP-E protein that interact with Mps1 protein. Methods: Two recombinant vectors named pEGFP- CENPE2 (containing 674-1085 amino acids of CENP-E protein) and pEGFP-CENPE 3(containing 1200~2134 amino acids of CENP-E protein) were transfected into human embryo kidney 293 (HEK293) cells respectively. The respective energy transfer efficiency (Ef) between either EGFP-CENPE2 and Mps1, or EGFP-CENPE3 and Mps1 were detected by FRET through selective photobleaching of the acceptors. Results: Both recombinant proteins expressed in HEK293 cells transfected by the recombinant plasmids were found to co-localize with the Mps1 protein as confirmed by confocal microscopy. The Ef between EGFP-CENPE3 and Mps1 protein was ［(12.63±0.48)%, n=30］ and that between EGFP-CENPE3 and Mps1 protein was ［(3.17±0.21)%, n=30］ as revealed by the results from FRET，the result of FRET was confirmed by coImmunoprecipitate(CO-IP) method. When compared with that between the control and Mps1, the Ef between EGFP-CENPE3 and Mps1 was significantly higher（p<0.05）, The results of CO-I Pshowed that EGFP-CENPE3 could interact with Mps1 protein. Conclusion: There was interaction between EGFP-CENPE3 and Mps1 as was demonstrated by the methods of FRET and CO-IP.

Key words: CENP-E;Mps1;protein-protein interaction;fluorescence resonance energy transfer

收稿日期: 2008-12-29 出版日期: 2009-04-27

ZTFLH:

Q71

基金资助:

国家自然科学基金

通讯作者: 翁亚光

	服务
	把本文推荐给朋友
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	刘子杰翁亚光李素彦施琼蔡燕刘斌张燕阎琛

引用本文:

刘子杰,翁亚光,李素彦,施琼,蔡燕,刘斌,张燕,阎琛. 用FRET方法研究与Mps1蛋白有相互作用的CENP-E蛋白结构域[J]. 中国生物工程杂志, 2009, 29(04): 28-34.

刘子杰,翁亚光,李素彦,施琼,蔡燕,刘斌,张燕,阎琛. Explore the Structural Domains Of CENP-E Protein Interacting with Mps1 protein by FRET method. China Biotechnology, 2009, 29(04): 28-34.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2009/V29/I04/28

［1］ May K M, Hardwick K G. The spindle checkpoint. J Cell Sci, 2006,119(Pt 20):4139~4142 ［2］ Kops G J. The kinetochore and spindle checkpoint in mammals. Front Biosci, 2008,13:3606~3620 ［3］ Taylor S S, Scott M I, Holland A J. The spindle checkpoint: a quality control mechanism which ensures accurate chromosome segregation. Chromosome Res, 2004,12(6):599~616 ［4］ Vigneron S, Prieto S, Bernis C, et al. Kinetochore localization of spindle checkpoint proteins: who controls whom?. Mol Biol Cell, 2004,15(10):4584~4596 ［5］张洁, 符传孩, 缪勇,等. 蛋白激酶TTK 与CENP-E 相互作用并共定位于人细胞的动粒. 科学通报, 2002,47(19): 1480~1484 Zhang J, Fu C H, Miao Y，et al.Chinese Science Bulletin，2002,47(19): 1480~1484 ［6］ GarciaSaez I, Yen T, Wade R H, et al. Crystal structure of the motor domain of the human kinetochore protein CENP-E. J Mol Biol, 2004,340(5):1107~1116 ［7］ Liu D, Zhang N, Du J, et al. Interaction of Skp1 with CENPE at the midbody is essential for cytokinesis. Biochem Biophys Res Commun, 2006,345(1):394~402 ［8］ Brown K D, Wood K W, Cleveland D W. The kinesin-like protein CENP-E is kinetochore-associated throughout poleward chromosome segregation during anaphase-A. J Cell Sci, 1996,109 ( Pt 5):961~969 ［9］ Karpova T S, Baumann C T, He L, et al. Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser. J Microsc, 2003,209(Pt 1):56~70 ［10］ Lleres D, Swift S, Lamond A I. Detecting protein-protein interactions in vivo with FRET using multiphoton fluorescence lifetime imaging microscopy (FLIM). Curr Protoc Cytom, 2007,Chapter 12:Unit12.10 ［11］ Stucke V M, Baumann C, Nigg E A. Kinetochore localization and microtubule interaction of the human spindle checkpoint kinase Mps1. Chromosoma, 2004,113(1):1~15 ［12］ Mao Y, Abrieu A, Cleveland D W. Activating and silencing the mitotic checkpoint through CENP-E-dependent activation/inactivation of BubR1. Cell, 2003,114(1):87~98 ［13］ Mao Y, Desai A, Cleveland D W. Microtubule capture by CENP-E silences BubR1dependent mitotic checkpoint signaling. J Cell Biol, 2005,170(6):873~880 ［14］ Chan G K, Jablonski S A, Sudakin V, et al. Human BUBR1 is a mitotic checkpoint kinase that monitors CENP-E functions at kinetochores and binds the cyclosome/APC. J Cell Biol, 1999,146(5):941~954 ［15］ Fisk H A, Mattison C P, Winey M. A field guide to the Mps1 family of protein kinases. Cell Cycle, 2004,3(4):439~442 ［16］ Espeut J, Gaussen A, Bieling P, et al. Phosphorylation relieves autoinhibition of the kinetochore motor CenpE. Mol Cell, 2008,29(5):637~643 ［17］ Abrieu A, MagnaghiJaulin L, Kahana J A, et al. Mps1 is a kinetochoreassociated kinase essential for the vertebrate mitotic checkpoint. Cell, 2001,106(1):83~93 ［18］ Chan G K, Schaar B T, Yen T J. Characterization of the kinetochore binding domain of CENP-E reveals interactions with the kinetochore proteins CENP-F and hBUBR1. J Cell Biol, 1998,143(1):49~63

[1]	易善勇, 杨晶, 官丽莉, 王艳芳, 黄建, 王立勇, 李海燕, 李校堃, 姜潮. 成纤维细胞生长因子9(FGF9)的研究进展[J]. 中国生物工程杂志, 2015, 35(7): 94-101.
[2]	张晨晨, 孟志忠, 陆远芳, 陈新, 李杉. 脱氮硫杆菌硫化合物载体SoxYZ蛋白的同源建模和结构分析[J]. 中国生物工程杂志, 2015, 35(7): 68-75.
[3]	刘冬梅, 孙佳楠, 邹佳凝, 刘明伟, 孙璐, 刘琼明. GSTpull-down联合质谱鉴定BAG结构域相互作用蛋白[J]. 中国生物工程杂志, 2015, 35(4): 1-10.
[4]	魏金梅, 范小琴, 熊海庭, 高学娟, 刘小会, 刘朗夏. hnRNPK与Nef相互作用并有利于细胞表面CD4的表达[J]. 中国生物工程杂志, 2015, 35(4): 17-22.
[5]	高星杰, 张毅, 苏超, 付雪, 史雪彬, 尹洁, 何津岩, 王鑫廷, 姚智, 杨洁. 针对人Tudor-SN蛋白T103位点的应激磷酸化抗体制备及分析[J]. 中国生物工程杂志, 2014, 34(06): 55-60.
[6]	李伟. 重组PLZF蛋白锌指结构域的表达、提纯和活性分析[J]. 中国生物工程杂志, 2011, 31(11): 1-5.
[7]	林瑛, 周倩, 荀启静, 孟清. 高剪接活性断裂蛋白质内含子的体内切割[J]. 中国生物工程杂志, 2011, 31(8): 97-101.
[8]	颜秋霞,吴晓萍,黄慧贤,聂昌君,聂燕芳,柯实,肖健,李校堃. bFGF特异性结合噬菌体的活性研究[J]. 中国生物工程杂志, 2009, 29(01): 23-26.
[9]	包士中,史晶,蔡昆,荫俊,王慧. Ⅱ型志贺样毒素B亚单位的重组表达及其受体结合活性[J]. 中国生物工程杂志, 2008, 28(10): 23-27.
[10]	尤晓娟,刘劲松. 重组人SNX9蛋白在大肠杆菌中的表达、纯化及性质分析[J]. 中国生物工程杂志, 2008, 28(8): 11-17.
[11]	郑玉姝,赵朴,刘兴友. 特异性miRNA调节机理研究进展[J]. 中国生物工程杂志, 2008, 28(2): 106-109.

Viewed

Full text

Abstract

Cited

Shared

Discussed