The Role Of WAC In Cell Cycle Regulation Through Activating Plk1

Polo-like kinase 1(Plk1),as a pivotal regulator of cell cycle,plays a key role in many steps of mitotic cell division ranging from mitotic entry to cytokinesis.During G2 phase,the kinase Aurora A(AurkA)cooperates with its co-activator Bora to phosphorylate Plk1 at Thr-210 and activate Plk1.Several lines of evidence suggest that Plk1 is activated at the centrosome.But,Plk1 activity is first seen in the nucleus.However,Bora is restricted to the cytoplasm in interphase of human cells,and is largely degraded during mitosis.It is unknown whether Bora-independent mechanisms of Plk1 activation exist in the nucleus of G2 cells.Taken together,the mechanism for Plk1 activation remains incompletely understood.In our quantitative phospho-proteomics analysis,we noticed WAC,a WW domain-containing adaptor protein with coiled-coil region,which was phosphorylated at multiple serine and threonine residues within highly conserved S-S/T-P motifs.Alignment of sequence shows that there exist five conserved S-S/T-P motifs in WAC.During G2 phase and mitosis,WAC undergoes Cdk1-dependent phosphorylation at its consensus S/T-P sites within multiple S-T-P motifs,and that WAC can be directly phosphorylated at these sites by Cdk1 in vitro.The results of GST pulldown assays,Far-western and co-immunoprecipitation experiments indicate that phosphorylation of WAC at these S-S/T-P motifs by Cdkl primes WAC binding to the PBD of Plkl.Knockdown of WAC causes compromised Plkl activity,delayed mitotic entry and impaired chromosome congression.These defects are rescued by expression of wild-type WAC but not the Plkl-binding-deficient mutant.Moreover,WAC directly.binds AurkA,and importantly,addition of Cdkl-phosphorylated MBP-WAC,but not the MBP-WAC-5A mutant defective in binding Plkl,strongly enhanced phosphorylation of GST-Plkl at T210 by 6xHiS-AurkA in vitro.Taken together,the phosphorylation of WAC by Cdkl enchances its interaction with Plkl,and then promotes Plkl activation and timely mitotic entry.The pricise regulation of mitosis is required for genomic stability,this study identifies WAC as a new binding protein of Plkl,and reveals an important role in activating Plkl during the G2/M transition,which can help us better understand the molecular mechanism of mitosis.