Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over 英文参考文献.docVIP

Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over 英文参考文献.doc

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Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over 英文参考文献

QuantitativeKineticStudyoftheActin-BundlingProtein L-PlastinandofItsImpactonActinTurn-Over ZiadAlTanoury1,ElisabethSchaffner-Reckinger1,AliaksandrHalavatyi1,Ce′lineHoffmann2,Miche`le Moes1,ErminHadzic1,MarieCatillon1,MikalaiYatskou1,EvelyneFriederich1* 1LaboratoryofCytoskeletonandCellPlasticity,LifeSciencesResearchUnit,UniversityofLuxembourg,LuxembourgCity,Luxembourg,2LaboratoryofPlantMolecular Biology,PublicResearchCenterforHealth(CRP-Sante′),Strassen,Luxembourg Abstract Background:Initiallydetectedinleukocytesandcancercellsderivedfromsolidtissues,L-plastin/fimbrinbelongstoalarge familyofactincrosslinkersandisconsideredasamarkerformanycancers.PhosphorylationofL-plastinonresidueSer5 increasesitsF-actinbindingactivityandisrequiredforL-plastin-mediatedcellinvasion. Methodology/PrincipalFindings:TostudythekineticsofL-plastinandtheimpactofL-plastinSer5phosphorylationonL- plastindynamicsandactinturn-overinlivecells,simianVerocellsweretransfectedwithGFP-coupledWT-L-plastin,Ser5 substitutionvariants(S5/A,S5/E)oractinandanalyzedbyfluorescencerecoveryafterphotobleaching(FRAP).FRAPdata wereexploredbymathematicalmodelingtoestimatesteady-statereactionparameters.WedemonstratethatinVerocell focal adhesions L-plastin undergoes rapid cycles of association/dissociation following a two-binding-state model. Phosphorylation of L-plastin increased its association rates by two-fold, whereas dissociation rates were unaffected. Importantly,L-plastinaffectedactinturn-overbydecreasingtheactindissociationratebyfour-fold,increasingtherebythe amount of F-actin in the focal adhesions, all these effects being promoted by Ser5 phosphorylation. In MCF-7 breast carcinoma cells, phorbol 12-myristate 13-acetate (PMA) treatment induced L-plastin translocation to de novo actin polymerization sites in ruffling membranes and spike-like structures and highly increased its Ser5 phosphorylation. Both inhibitionstudiesandsiRNAknock-downofPKCisozymespointedtotheinvolvementofthenovelPKC-disozymeinthe PMA-el

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