Molecular Mechanisms Of14-3-3Cooperates With Plk1to Regulate Cytokinesis

Proteins of the14-3-3family are functionally conserved in eukaryotic kingdom whichparticipates in diversified and critical cellular processes. However, the exact roles ofthese proteins in mitotic regulation has remained elusive. Polo-like kinase1（Plk1） is aserine/threonine protein kinase that plays multiple critical functions such as centrosomematuration, mitotic chromosome segregation, cytokinesis, and the DNA damageresponse. Here we show that14-3-3zeta interacts and cooperates with Plk1in mitoticprogress.14-3-3zeta is associated with the spindle at metaphase and concentrated in themidbody during cytokinesis.Using yeast two hybrid assay, we found a functionalconnection between14-3-3zeta and Plk1. We demonstrate that phosphorylation of Plk1at S330and S597promotes its interaction with14-3-3zeta.Importantly,14-3-3zetacooperates with Plk1in ensuring successful cytokinesis. We conclude that mitoticphosphorylation of Plk1promotes interaction with14-3-3zeta and this interaction isrequired for faithful cytokinesis. Taken together with the results of previous studies, our results suggest14-3-3family emerges as a novel player in mitotic regulation:cooperation with Plk1to ensure a faithful cytokinesis.Objective：To screen14-3-3zeta-interacting proteins by yeast two-hybrid system,confirm theinteraction between14-3-3zeta and Plk1and investigate whether the interactionbetween14-3-3zeta and Plk1is required for faithful cytokinesis.Methods:Immunofluorescence staining investigate the subcellular distribution of14-3-3isoforms in HeLa cells. Identifying the interaction between14-3-3zeta and Plk1byyeast two-hybrid screening or GST pull-down assay or co-immunoprecipitation assay.To test whether phosphorylated Plk1at S330/597specifically interacts with14-3-3zeta,constructs consisting of wild type Plk1（Plk1-WT）, phospho-mimicking mutant Plk1(Plk1^S330/597D) and non-phosphorylatable mutants Plk1（Plk1S330/597A） were fused toGFP.Knock down14-3-3zeta or ectopic express the Plk1phosphorylation mutantsto examine whether the interaction between14-3-3ζ and Plk1ensure a faithfulcytokinesis.Results:1.14-3-3zeta is associated with the spindle at metaphase and concentrates in themidbody during cytokinesisSynchronized HeLa cells were extracted in PHEM buffer containing0.1%Triton X-100at room temperature,fixed in4%formaldehyde in PHEM buffer for10min.Opticalimages collected from one HeLa cell stained for14-3-3isforms with specific antibodies（beta, gamma, sigma, epsilon, zeta and eta）（14-3-3isforms, green）,DAPI（DNA,blue）and their merged images. The results show that14-3-3zeta is associated with the spindle at metaphase and concentrates in the midbody during cytokinesis.2.14-3-3zeta interacts with Plk12.1Yeast two-hybrid screeningTo better understand the role of14-3-3zeta in cellular dynamics, we performed a yeasttwo-hybrid to screen a human HeLa library using14-3-3zeta as the bait.Among thepositive clones, one interesting candidate is Polo-like Kinase1（Plk1）.2.2Co-transformation AssayAH109cells were co-transformed pGADT7-Plk1and pGBKT7-14-3-3zeta and thenselected on supplemented minimal plates lacking tryptophan, leucine, histidine,andadenine. The interaction between Plk1and14-3-3zeta was revealed through staining forbeta-galactosidase activity with X-α-Gal. The co-transformation assay confirmed thatPlk1interacts with14-3-3zeta.2.3ImmunofluorescenceSynchronized HeLa cells were extracted, fixed and stained with rabbit anti-14-3-3zetaantibody（green）,mouse anti-Plk1antibody （red）,DAPI （blue）.The merged images showthat14-3-3zeta colocalized with Plk1at the midbody.2.4Co-immunoprecipitation Assay2.4.1Exogenous Co-immunoprecipitation AssayHEK293T cells were transfected FLAG-tagged Plk1with GFP tagged wild type14-3-3zeta or GFP tagged14-3-3zeta K49E or GFP. FLAG-tagged Plk1protein and itsaccessory were precipitated with10μl FLAG-M2-antibody-conjugated beads.Coprecipitated proteins were then immunoblotted for the presence of GFP-14-3-3zetaWT, the binding null14-3-3zetaK49E mutant proteins and GFP proteins or FLAG-Plk1.The results show that the interaction between Plk1and14-3-3zeta also occurs inmammalian cells.2.4.2Endogenous Co-immunoprecipitation AssayLysates from interphase and mitotic HeLa cells were incubated with an antibody against 14-3-3zeta and the immunoprecipitates were examined for the presence ofcoprecipitating Plk1. IP obtained with14-3-3zeta antibodies were found to contain14-3-3zeta as well as co-precipitating Plk1during mitotic cell. Our result also showedthe interaction between endogenous14-3-3zeta and Plk1occurs specifically duringmitosis because the interaction level between14-3-3zeta and Plk1during interphase cellis very low.2.5GST-pull down AssayTo test the direct binding between Plk1and14-3-3, we carried out a pull-down assay inwhich histidine-tagged recombinant Plk1was purified on Ni-NTA agarose beads andused as an affinity matrix to absorb purified GST-14-3-3zeta in test tubes. However,GST-tagged14-3-3protein failed to pull down Plk1recombinant protein.3. Phosphorylation of Plk1at S330/597Enhances14-3-3zeta Binding3.1The interaction between14-3-3zeta and Plk1is phosphorylate dependentWe transiently transfected Flag-Plk1into HEK293T cells, then treated cell lysate withthe serine/threonine phosphatase inhibitor okadaic acid or λ-phosphatase. Cell lysateswere then subjected to incubation with GST or GST-fused14-3-3zeta. Western blotanalysis of both control and treated samples revealed that a high amount of Plk1wasretained by the GST-14-3-3zeta while treatment with okadaic acid.3.2Phosphorylation of Plk1at S330/597enhances14-3-3zeta bindingTo test whether phosphorylated Plk1at S330/597specifically interacts with14-3-3zeta,constructs consisting of wild type Plk1（Plk1-WT）, phospho-mimicking mutantPlk1(Plk1^S330/597D) and non-phosphorylatable mutants Plk1（Plk1S330/597A） were fused toGFP. We transiently transfected the above GFP-tagged constructs into HEK293T. Celllysates were then subjected to incubation with glutathione agarose tagged with GST orwith the GSTfused14-3-3zeta.The results of Western analysis indicate that asignificant amount of Plk1^S330/597Dprotein was retained by the GST-14-3-3zeta.Toevaluate the phosphor-regulation of Plk1and14-3-3zeta interaction, we used a GST pull down assay in vitro. GST fused14-3-3zeta recombinant protein was purified onglutathione-agarose beads and used as affinity matrix for absorbing histidine-taggedwild type Plk1and histidine-tagged phosphor-mimicking mutant Plk1^S330/597Dasdescribed in experimental procedure. Glutathione-agarose beads pre-bound GST proteinwas used as a negative control.The results of coomassie blue staining and western-blotshow that phosphor-mimicking mutant Plk1^S330/597Dbinds to14-3-3zeta while theinteraction level between wild type Plk1and14-3-3zeta is very low.3.3The interaction between phosphorylated Plk1at S330/597and14-3-3zetaoccurs in mitotic HeLa cellsWe used anti-FLAG antibodies to immunoprecipitate soluble14-3-3zeta and itsbinding proteins from lysates of mitotic arrested HeLa cells transiently transfected toexpress GFP-Plk1-WT, GFP-Plk1^S330/597Dand GFP-Plk1S330/597Arespectively. Westernblot with GFP antibody validated that a significant amount of Plk1^S330/597Dprotein iscoprecipitated with14-3-3zeta.However, the interaction level between wild type Plk1and14-3-3zeta was very low. No GFP-Plk1S330/597Awas recovered in the14-3-3zetaimmunoprecipitates.4.14-3-3zeta cooperates with Plk1to complete correct cytokinesis4.1Plk1is essential for the mitotic localization of14-3-3zetaHeLa cells were transfected with control siRNA,14-3-3siRNA, and Plk1siRNA,synchronized HeLa cells were extracted, fixed and stained with rabbit anti-14-3-3zetaantibody（green）,mouse anti-Plk1antibody （red）,DAPI （blue）.The merged images showthat no significant effects on Plk1localization were observed in response to14-3-3zetadepletion. However, the spindle and midbody localization of14-3-3zeta was diminishedwhen Plk1protein expression was repressed, suggesting that Plk1is essential for themitotic localization of14-3-3zeta.4.2Knock down endogenous14-3-3zeta resulted in significant cytokinesis defectsWe investigated the role of14-3-3zeta in mitosis by depletion of endogenous14-3-3zeta with siRNA in HeLa cells. We found that downregulation of endogenous14-3-3zetaresulted in a population of cells arrested at midbody stage and polyploidy.Control-siRNA treated cells showed no significant cytokinesis defect. Examination of14-3-3zeta siRNA treated cells at various times showed that the percentage ofmultinucleated cells continued to increase after72hr, whereas the percentage of cells atmidbody stage peaked at72hr post-transfection.4.3Ectopic Expression of the Plk1Phosphorylation Mutants Induces CytokinesisFailureHeLa cells were transiently transfected to express GFP-Plk1-WT, GFP-Plk1^S330/597Dand GFP-Plk1S330/597Arespectively and then extracted, fixed and stained.The resultesshow that phosphorylation of Plk1at S330/597is essential for correct cytokinesis.Conclusions:14-3-3zeta cooperates with Plk1to complete correct cytokinesis.