Polo-like kinase 1 (Plk1) is one of the major kinases controlling mitosis and cell division. Plk1 is first recruited to the centrosome in S phase, then appears on the kinetochores in late G2, and at the end of mitosis, it translocates to the central spindle. Activation of Plk1 requires phosphorylation of T210 by Aurora A, an event that critically depends on the co-factor Bora. However, conflicting reports exist as to where Plk1 is first activated. Phosphorylation of T210 is first observed at the centrosomes, but kinase activity seems to be restricted to the nucleus in the earlier phases of G2. Here, we demonstrate that Plk1 activity manifests itself first in the nucleus using a nuclear FRET-based biosensor for Plk1 activity. However, we find that Bora is restricted to the cytoplasm and that Plk1 is phosphorylated on T210 at the centrosomes. Our data demonstrate that while Plk1 activation occurs on centrosomes, downstream target phosphorylation by Plk1 first occurs in the nucleus. We discuss several explanations for this surprising separation of activation and function.

Department of Cell Biology The Netherlands Cancer Institute Amsterdam Netherlands ; Department of Medical Oncology and Cancer Genomics Center University Medical Center Utrecht Utrecht Netherlands

Department of Medical Oncology and Cancer Genomics Center University Medical Center Utrecht Utrecht Netherlands

Department of Medical Oncology and Cancer Genomics Center University Medical Center Utrecht Utrecht Netherlands ; Department of Cell and Molecular Biology Karolinska Institute Stockholm Sweden

Department of Medical Oncology and Cancer Genomics Center University Medical Center Utrecht Utrecht Netherlands ; Laboratory of Cancer Cell Biology Institute of Molecular Genetics of the ASCR v v i Prague Czech Republic

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Bruinsma W, Raaijmakers JA, Medema RH. Switching Polo-like kinase-1 on and off in time and space. Trends Biochem Sci (2012) 37:534–42.10.1016/j.tibs.2012.09.005 PubMed DOI

Petronczki M, Glotzer M, Kraut N, Peters J-M. Polo-like kinase 1 triggers the initiation of cytokinesis in human cells by promoting recruitment of the rhogef ect2 to the central spindle. Dev Cell (2007) 12:713–25.10.1016/j.devcel.2007.03.013 PubMed DOI

Elia AEH, Cantley LC, Yaffe MB. Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates. Science (2003) 299:1228–31.10.1126/science.1079079 PubMed DOI

Elia AEH, Rellos P, Haire LF, Chao JW, Ivins FJ, Hoepker K, et al. The molecular basis for phosphodependent substrate targeting and regulation of Plks by the Polo-box domain. Cell (2003) 115:83–95.10.1016/S0092-8674(03)00725-6 PubMed DOI

Kang YH, Park J-E, Yu L-R, Soung N-K, Yun S-M, Bang JK, et al. Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for proper chromosome segregation. Mol Cell (2006) 24:409–22.10.1016/j.molcel.2006.10.016 PubMed DOI

Leung GC, Hudson JW, Kozarova A, Davidson A, Dennis JW, Sicheri F. The Sak Polo-box comprises a structural domain sufficient for mitotic subcellular localization. Nat Struct Biol (2002) 9:719–24.10.1038/nsb848 PubMed DOI

Neef R, Gruneberg U, Kopajtich R, Li X, Nigg EA, Sillje H, et al. Choice of Plk1 docking partners during mitosis and cytokinesis is controlled by the activation state of Cdk1. Nat Cell Biol (2007) 9:436–44.10.1038/ncb1557 PubMed DOI

Qi W, Tang Z, Yu H. Phosphorylation- and Polo-box-dependent binding of Plk1 to Bub1 is required for the kinetochore localization of Plk1. Mol Biol Cell (2006) 17:3705–16.10.1091/mbc.E06-03-0240 PubMed DOI PMC

Soung N-K, Park J-E, Yu L-R, Lee KH, Lee J-M, Bang JK, et al. Plk1-dependent and -independent roles of an ODF2 splice variant, hCenexin1, at the centrosome of somatic cells. Dev Cell (2009) 16:539–50.10.1016/j.devcel.2009.02.004 PubMed DOI PMC

Jang Y-J, Ma S, Terada Y, Erikson RL. Phosphorylation of threonine 210 and the role of serine 137 in the regulation of mammalian polo-like kinase. J Biol Chem (2002) 277:44115–20.10.1074/jbc.M202172200 PubMed DOI

Seki A, Coppinger JA, Jang C-Y, Yates JR, Fang G. Bora and the kinase aurora a cooperatively activate the kinase Plk1 and control mitotic entry. Science (2008) 320:1655–8.10.1126/science.1157425 PubMed DOI PMC

Macurek L, Lindqvist A, Lim D, Lampson MA, Klompmaker R, Freire R, et al. Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery. Nature (2008) 455:119–23.10.1038/nature07185 PubMed DOI

Barr AR, Gergely F. Aurora-A: the maker and breaker of spindle poles. J Cell Sci (2007) 120(Pt 17):2987–96.10.1242/jcs.013136 PubMed DOI

Bruinsma W, Macurek L, Freire R, Lindqvist A, Medema RH. Bora and Aurora-A continue to activate Plk1 in mitosis. J Cell Sci (2014) 127(Pt 4):801–11.10.1242/jcs.137216 PubMed DOI

Akopyan K, Silva Cascales H, Hukasova E, Saurin AT, Müllers E, Jaiswal H, et al. Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition. Mol Cell (2014) 53:843–53.10.1016/j.molcel.2014.01.031 PubMed DOI

Hutterer A, Berdnik D, Wirtz-Peitz F, Zigman M, Schleiffer A, Knoblich JA. Mitotic activation of the kinase aurora-A requires its binding partner Bora. Dev Cell (2006) 11:147–57.10.1016/j.devcel.2006.06.002 PubMed DOI

Feine O, Hukasova E, Bruinsma W, Freire R, Fainsod A, Gannon J, et al. Phosphorylation-mediated stabilization of Bora in mitosis coordinates Plx1/Plk1 and Cdk1 oscillations. Cell Cycle (2014) 13(11):1727–36.10.4161/cc.28630 PubMed DOI PMC

Chan EHY, Santamaria A, Sillje HHW, Nigg EA. Plk1 regulates mitotic aurora A function through betaTrCP-dependent degradation of hBora. Chromosoma (2008) 117:457–69.10.1007/s00412-008-0165-5 PubMed DOI PMC

Seki A, Coppinger JA, Du H, Jang C-Y, Yates JR, Fang G. Plk1- and beta-TrCP-dependent degradation of Bora controls mitotic progression. J Cell Biol (2008) 181:65–78.10.1083/jcb.200712027 PubMed DOI PMC

Hukasova E, Silva Cascales H, Kumar SR, Lindqvist A. Monitoring kinase and phosphatase activities through the cell cycle by ratiometric FRET. J Vis Exp (2012) (59):e3410.10.3791/3410 PubMed DOI PMC

Méndez J, Stillman B. Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol Cell Biol (2000) 20(22):8602–12.10.1128/MCB.20.22.8602-8612.2000 PubMed DOI PMC

Lénárt P, Petronczki M, Steegmaier M, Di Fiore B, Lipp JJ, Hoffmann M, et al. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1. Curr Biol (2007) 17:304–15.10.1016/j.cub.2006.12.046 PubMed DOI

Manfredi MG, Ecsedy JA, Meetze KA, Balani SK, Burenkova O, Chen W, et al. Antitumor activity of MLN8054, an orally active small-molecule inhibitor of aurora A kinase. Proc Natl Acad Sci U S A (2007) 104:4106–11.10.1073/pnas.0608798104 PubMed DOI PMC

Cheeseman IM, Desai A. A combined approach for the localization and tandem affinity purification of protein complexes from metazoans. Sci STKE (2005) 2005:l1.10.1126/stke.2662005pl1 PubMed DOI

Gillingham AK, Munro S. The PACT domain, a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin. EMBO Rep (2000) 1:524–9.10.1093/embo-reports/kvd105 PubMed DOI PMC

van Vugt MATM, Brás A, Medema RH. Polo-like kinase-1 controls recovery from a G2 DNA damage-induced arrest in mammalian cells. Mol Cell (2004) 15:799–811.10.1016/j.molcel.2004.07.015 PubMed DOI

Takaki T, Trenz K, Costanzo V, Petronczki M. Polo-like kinase 1 reaches beyond mitosis – cytokinesis, DNA damage response, and development. Curr Opin Cell Biol (2008) 20:650–60.10.1016/j.ceb.2008.10.005 PubMed DOI

Rannou Y, Troadec M-B, Petretti C, Hans F, Duterte S, Dimitrov S, et al. Localization of aurora A and aurora B kinases during interphase. Cell Cycle (2008) 7:3012–20.10.4161/cc.7.19.6718 PubMed DOI PMC

Alvarez-Fernández M, Sánchez-Martínez R, Sanz-Castillo B, Gan PP, Sanz-Flores M, Trakala M, et al. Greatwall is essential to prevent mitotic collapse after nuclear envelope breakdown in mammals. Proc Natl Acad Sci U S A (2013) 110:17374–9.10.1073/pnas.1310745110 PubMed DOI PMC

Wang P, Galan JA, Normandin K, Bonneil É, Hickson GR, Roux PP, et al. Cell cycle regulation of greatwall kinase nuclear localization facilitates mitotic progression. J Cell Biol (2013) 202:277–93.10.1083/jcb.201211141 PubMed DOI PMC

Mochida S, Maslen SL, Skehel M, Hunt T. Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis. Science (2010) 330:1670–3.10.1126/science.1195689 PubMed DOI

Wang L, Guo Q, Fisher LA, Liu D, Peng A. Regulation of polo-like kinase 1 by DNA damage and PP2A/B55α. Cell Cycle (2015) 14:157–66.10.4161/15384101.2014.986392 PubMed DOI PMC

Kishi K, van Vugt MATM, Okamoto K-I, Hayashi Y, Yaffe MB. Functional dynamics of Polo-like kinase 1 at the centrosome. Mol Cell Biol (2009) 29:3134–50.10.1128/MCB.01663-08 PubMed DOI PMC

Goto H, Kiyono T, Tomono Y, Kawajiri A, Urano T, Furukawa K, et al. Complex formation of Plk1 and INCENP required for metaphase–anaphase transition. Nat Cell Biol (2005) 8:180–7.10.1038/ncb1350 PubMed DOI

Elowe S, Hümmer S, Uldschmid A, Li X, Nigg EA. Tension-sensitive Plk1 phosphorylation on BubR1 regulates the stability of kinetochore microtubule interactions. Genes Dev (2007) 21:2205–19.10.1101/gad.436007 PubMed DOI PMC

Hirota T, Kunitoku N, Sasayama T, Marumoto T, Zhang D, Nitta M, et al. Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells. Cell (2003) 114:585–98.10.1016/S0092-8674(03)00642-1 PubMed DOI

Joukov V, Walter JC, De Nicolo A. The Cep192-Organized Aurora A-Plk1 cascade is essential for centrosome cycle and bipolar spindle assembly. Mol Cell (2014) 55:578–91.10.1016/j.molcel.2014.06.016 PubMed DOI PMC

Multiple Roles of PLK1 in Mitosis and Meiosis

Cyclin A2 localises in the cytoplasm at the S/G2 transition to activate PLK1