为了鉴定hHBRK1的相互作用蛋白,通过DNA重组构建重组表达质粒pGEXhHBRK1,并以谷胱甘肽Sepharose4B亲合层析法,获得纯化的重组融合蛋白GSThHBRK1.以小鼠心肌组织为研究对象,采用GSTpulldown技术结合Western印迹法,证实hHBRK1与小鼠心肌肌钙蛋白TEa亚型(EacTnT)相互作用.结果提示,hHBRK1与EacTnT结合,可能参与心肌微丝的聚合,为小鼠cTnT众多的剪接体,提供了一种可能的功能定位.
Abstract
Brick 1 gene(Brk 1),highly conserved in plants and animals,plays important role in reorganization of cortical actin filaments during cell movement and pattern formation.To characterize the interacting protein for hHBRK1, the coding region of hHBrk1 was isolated and subcloned into plasmid pGEX-4T fused in frame to generate pGEX-hHBrk1. GST-hHBRK1 fusion protein was purified from bacterial lysates using glutathion-Sepharose 4B affinity chromatography. For GST pull-down assay, the heart tissue of mouse was selected to be positive control. The interaction between hHBRK1 and Ea isoform of cardiac troponin T (Ea-cTnT) was unveiled by GST pull-down assay followed by peptide finger mass spectrometry and further confirmed by Western blotting. These results suggested that hHBRK1 binded Ea isoform of cTnT and the N-terminus of cTnT might be the region where the interaction takes place, hHBRK1-cTnT interaction might regulate actin cytoskeletal organization.
关键词
BRK1 /
肌钙蛋白 /
肌动蛋白 /
鉴定
{{custom_keyword}} /
Key words
BRK1 /
troponin T /
actin /
characterization
{{custom_keyword}} /
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
1 徐勤枝,丁新民,段瑞峰,周平坤,项晓琼,张开泰,吴德昌.微丝相关新基因hHBrk1的克隆及功能鉴定.中国生物化学与分子生物学报(XuQin Zhi,DingXin Min,DuanRui Feng,ZhouPing Kun,XiangXiao Qiong,ZhangKai Tai,WuDe Chang.Cloningand identificationofanovelgenehHBrk1,encodingaF actinassociated proteinhighlyconservedinanimalsandplants.ChinJBiochemMolBiol),2004,20(5):610～614
2 EdenS,RohatgiR,PodtelejnikovAV,MannM,KirschnerMW.MechanismofregulationofWAVE1inducedactinnucleationbyRac1andNck.Nature,2002,418:790～793
3 FrankMJ,SmithLG.Asmall,novelproteinhighlyconservedin plantsandanimalspromotesthepolarizedgrowthanddivisionofmaize leafepidermalcells.CurrBiol,2002,12:849～853
4 FrankMJ,CartwrightHN,SmithLG.Threebrickgeneshavedistinct functionsinacommonpathwaypromotingpolarizedcelldivisionandcell morphogenesisinthemaizeleafepidermis.Development,2003,130:753～762
5 SambrookJ,RussellDW.MolecularCloning:ALaboratoryManual,3rded.NewYork:ColdSpringHarborLaboratoryPress,2001
6 PawsonT,NashP.Protein proteininteractionsdefinespecificityin signaltransduction.GenesDev,2000,14:1027～1047
7 TobacmanLS.Thinfilament mediatedregulationofcardiac contraction.AnnuRevPhysiol,1996,58:4474～4481
8 HernandezOM,HousmansPR,PotterJD.Pathophysiologyofcardiac musclecontractionandrelaxationasaresultofalterationsinthin filamentregulation.JApplPhysiol,2001,90:1125～1136
9 HinkleA,GoransonA,ButtersCA,TobacmanLS.Rolesforthe troponintaildomaininthinfilamentassemblyandregulation.A deletionalstudyofcardiactroponinT.JBiolChem,1999,274:7157～7164
10 CramerLP.MyosinⅥ:rolesforaminusend directedactinmotorin cells.JCellBiol,2000,150:F121～126
11 BussF,Kendrick JonesJ,LionneC,KnightAE,CoteGP,LuzioJ P.ThelocalizationofmyosinⅥatthegolgicomplexandleadingedge offibroblastsanditsphosphorylationandrecruitmentintomembrane rufflesofA431cellsaftergrowthfactorstimulation.JCellBiol,1998,143:1535～1545
12 RogatAD,MillerKG.AroleformyosinⅥinactindynamicsatsites ofmembraneremodelingduringDrosophilaspermatogenesis.JCellSci,2002,115(24):4855～4865
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
PDF(318 KB)
