Distinct Pools of cdc25C Are Phosphorylated on Specific TP Sites and Differentially Localized in Human Mitotic Cells 英文参考文献.docVIP

DistinctPoolsofcdc25CArePhosphorylatedonSpecific TPSitesandDifferentiallyLocalizedinHumanMitotic Cells CelineFranckhauser,DariaMamaeva,LisaHeron-Milhavet,AnneFernandez,NedJ.C.Lamb* CellBiologyUnit,InstitutedeGenetiqueHumain,CNRS-UPR1142,Montpellier,France Abstract Background:Thedualspecificityphosphatasecdc25Cwasthefirsthumancdc25familymemberfoundtobeessentialin the activation of cdk1/cyclin B1 that takes place at the entry into mitosis. Human cdc25C is phosphorylated on Proline- dependent SP and TP sites when it becomes active at mitosis and the prevalent model is that this phosphorylation/ activationofcdc25Cwouldbepartofanamplificationloopwithcdk1/cyclinB1. Methodology/Principal Findings: Using highly specific antibodies directed against cdc25C phospho-epitopes, pT67 and pT130,weshowherethatthesetwophospho-formsofcdc25Crepresentdistinctpoolswithdifferentiallocalizationduring humanmitosis.PhosphorylationonT67occursfromprophaseandthecdc25C-pT67phospho-isoformcloselylocalizeswith condensed chromosomes throughout mitosis. The phospho-T130 form of cdc25C arises in late G2 and associates predominantly with centrosomes from prophase to anaphase B where it colocalizes with Plk1. As shown by immunoprecipitation of each isoform, these two phospho-forms are not simultaneously phosphorylated on the other mitotic TP sites or associated with one another. Phospho-T67 cdc25C co-precipitates with MPM2-reactive proteins while pT130-cdc25C is associated with Plk1. Interaction and colocalization of phosphoT130-cdc25C with Plk1 demonstrate in livingcells,thatthesequencearoundpT130actsasatruePoloBoxDomain(PBD)bindingsiteaspreviouslyidentifiedfrom invitropeptidescreeningstudies.Overexpressionofnon-phosphorylatablealaninemutantformsforeachisoform,butnot wild type cdc25C, strongly impairs mitotic progression showing the functional requirement for each site-specific phosphorylationofcdc25Catmitosis. Conclusions/Significance:Theseresultsshowforthefirsttimethatinhumanmitosis,distinctphos