Cyclin A2 is a key regulator of the cell cycle, implicated both in DNA replication and mitotic entry. Cyclin A2 participates in feedback loops that activate mitotic kinases in G2 phase, but why active Cyclin A2-CDK2 during the S phase does not trigger mitotic kinase activation remains unclear. Here, we describe a change in localisation of Cyclin A2 from being only nuclear to both nuclear and cytoplasmic at the S/G2 border. We find that Cyclin A2-CDK2 can activate the mitotic kinase PLK1 through phosphorylation of Bora, and that only cytoplasmic Cyclin A2 interacts with Bora and PLK1. Expression of predominately cytoplasmic Cyclin A2 or phospho-mimicking PLK1 T210D can partially rescue a G2 arrest caused by Cyclin A2 depletion. Cytoplasmic presence of Cyclin A2 is restricted by p21, in particular after DNA damage. Cyclin A2 chromatin association during DNA replication and additional mechanisms contribute to Cyclin A2 localisation change in the G2 phase. We find no evidence that such mechanisms involve G2 feedback loops and suggest that cytoplasmic appearance of Cyclin A2 at the S/G2 transition functions as a trigger for mitotic kinase activation.

Department of Cell and Molecular Biology Karolinska Institutet Stockholm Sweden

Laboratory of Cancer Cell Biology Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Abdurashidova G, Deganuto M, Klima R, Riva S, Biamonti G, Giacca M, Falaschi A (2000) Start sites of bidirectional DNA synthesis at the human lamin B2 origin. Science 287: 2023–2026. 10.1126/science.287.5460.2023 PubMed DOI

Akopyan K, Lindqvist A, Mullers E (2016) Cell cycle dynamics of proteins and post-translational modifications using quantitative immunofluorescence. Methods Mol Biol 1342: 173–183. 10.1007/978-1-4939-2957-3_9 PubMed DOI

Akopyan K, Silva Cascales H, Hukasova E, Saurin AT, Mullers E, Jaiswal H, Hollman DA, Kops GJ, Medema RH, Lindqvist A (2014) Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition. Mol Cell 53: 843–853. 10.1016/j.molcel.2014.01.031 PubMed DOI

Andersen JS, Lyon CE, Fox AH, Leung AKL, Lam YW, Steen H, Mann M, Lamond AI (2002) Directed proteomic analysis of the human nucleolus. Curr Biol 12: 1–11. 10.1016/s0960-9822(01)00650-9 PubMed DOI

Arsic N, Bendris N, Peter M, Begon-Pescia C, Rebouissou C, Gadea G, Bouquier N, Bibeau F, Lemmers B, Blanchard JM (2012) A novel function for cyclin A2: Control of cell invasion via RhoA signaling. J Cell Biol 196: 147–162. 10.1083/jcb.201102085 PubMed DOI PMC

Baranello L, Wojtowicz D, Cui K, Devaiah BN, Chung HJ, Chan-Salis KY, Guha R, Wilson K, Zhang X, Zhang H, et al. (2016) RNA polymerase II regulates topoisomerase 1 activity to favor efficient transcription. Cell 165: 357–371. 10.1016/j.cell.2016.02.036 PubMed DOI PMC

Bendris N, Lemmers B, Blanchard JM, Arsic N (2011) Cyclin A2 mutagenesis analysis: A new insight into CDK activation and cellular localization requirements. PLoS One 6: e22879 10.1371/journal.pone.0022879 PubMed DOI PMC

Bruinsma W, Aprelia M, Kool J, Macurek L, Lindqvist A, Medema RH (2015) Spatial separation of Plk1 phosphorylation and activity. Front Oncol 5: 132 10.3389/fonc.2015.00132 PubMed DOI PMC

Bruinsma W, Macurek L, Freire R, Lindqvist A, Medema RH (2014) Bora and Aurora-A continue to activate Plk1 in mitosis. J Cell Sci 127: 801–811. 10.1242/jcs.137216 PubMed DOI

Cardoso MC, Leonhardt H, Nadal-Ginard B (1993) Reversal of terminal differentiation and control of DNA replication: Cyclin A and Cdk2 specifically localize at subnuclear sites of DNA replication. Cell 74: 979–992. 10.1016/0092-8674(93)90721-2 PubMed DOI

Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J, et al. (2006) CellProfiler: Image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7: R100 10.1186/gb-2006-7-10-r100 PubMed DOI PMC

Crncec A, Hochegger H (2019) Triggering mitosis. FEBS Lett 593: 2868–2888. 10.1002/1873-3468.13635 PubMed DOI

De Boer L, Oakes V, Beamish H, Giles N, Stevens F, Somodevilla-Torres M, Desouza C, Gabrielli B (2008) Cyclin A/cdk2 coordinates centrosomal and nuclear mitotic events. Oncogene 27: 4261–4268. 10.1038/onc.2008.74 PubMed DOI

Feine O, Hukasova E, Bruinsma W, Freire R, Fainsod A, Gannon J, Mahbubani HM, Lindqvist A, Brandeis M (2014) Phosphorylation-mediated stabilization of Bora in mitosis coordinates Plx1/Plk1 and Cdk1 oscillations. Cell Cycle 13: 1727–1736. 10.4161/cc.28630 PubMed DOI PMC

Frouin I, Toueille M, Ferrari E, Shevelev I, Hubscher U (2005) Phosphorylation of human DNA polymerase lambda by the cyclin-dependent kinase Cdk2/cyclin A complex is modulated by its association with proliferating cell nuclear antigen. Nucleic Acids Res 33: 5354–5361. 10.1093/nar/gki845 PubMed DOI PMC

Fung TK, Ma HT, Poon RY (2007) Specialized roles of the two mitotic cyclins in somatic cells: Cyclin A as an activator of M phase-promoting factor. Mol Biol Cell 18: 1861–1873. 10.1091/mbc.E06-12-1092 PubMed DOI PMC

Furuno N, den Elzen N, Pines J (1999) Human cyclin A is required for mitosis until mid prophase. J Cell Biol 147: 295–306. 10.1083/jcb.147.2.295 PubMed DOI PMC

Gheghiani L, Loew D, Lombard B, Mansfeld J, Gavet O (2017) PLK1 activation in late G2 sets up commitment to mitosis. Cell Rep 19: 2060–2073. 10.1016/j.celrep.2017.05.031 PubMed DOI

Gong D, Ferrell JE Jr. (2010) The roles of cyclin A2, B1, and B2 in early and late mitotic events. Mol Biol Cell 21: 3149–3161. 10.1091/mbc.E10-05-0393 PubMed DOI PMC

Gong D, Pomerening JR, Myers JW, Gustavsson C, Jones JT, Hahn AT, Meyer T, Ferrell JE Jr. (2007) Cyclin A2 regulates nuclear-envelope breakdown and the nuclear accumulation of cyclin B1. Curr Biol 17: 85–91. 10.1016/j.cub.2006.11.066 PubMed DOI PMC

Hégarat N, Crncec A, Rodriguez MFSP, Iturra FE, Gu Y, Busby O, Lang PF, Barr AR, Bakal C, Kanemaki MT, et al. (2020) Cyclin A triggers Mitosis either via the greatwall kinase pathway or Cyclin B. EMBO J 39: e104419 10.15252/embj.2020104419 PubMed DOI PMC

Hein JB, Nilsson J (2016) Interphase APC/C-Cdc20 inhibition by cyclin A2-Cdk2 ensures efficient mitotic entry. Nat Commun 7: 10975 10.1038/ncomms10975 PubMed DOI PMC

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

Jackman M, Kubota Y, den Elzen N, Hagting A, Pines J (2002) Cyclin A- and cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm. Mol Biol Cell 13: 1030–1045. 10.1091/mbc.01-07-0361 PubMed DOI PMC

Johmura Y, Shimada M, Misaki T, Naiki-Ito A, Miyoshi H, Motoyama N, Ohtani N, Hara E, Nakamura M, Morita A, et al. (2014) Necessary and sufficient role for a mitosis skip in senescence induction. Mol Cell 55: 73–84. 10.1016/j.molcel.2014.05.003 PubMed DOI

Kachaner D, Garrido D, Mehsen H, Normandin K, Lavoie H, Archambault V (2017) Coupling of Polo kinase activation to nuclear localization by a bifunctional NLS is required during mitotic entry. Nat Commun 8: 1701 10.1038/s41467-017-01876-8 PubMed DOI PMC

Kalaszczynska I, Geng Y, Iino T, Mizuno S, Choi Y, Kondratiuk I, Silver DP, Wolgemuth DJ, Akashi K, Sicinski P (2009) Cyclin A is redundant in fibroblasts but essential in hematopoietic and embryonic stem cells. Cell 138: 352–365. 10.1016/j.cell.2009.04.062 PubMed DOI PMC

Kanakkanthara A, Jeganathan KB, Limzerwala JF, Baker DJ, Hamada M, Nam HJ, van Deursen WH, Hamada N, Naylor RM, Becker NA, et al. (2016) Cyclin A2 is an RNA binding protein that controls Mre11 mRNA translation. Science 353: 1549–1552. 10.1126/science.aaf7463 PubMed DOI PMC

Katsuno Y, Suzuki A, Sugimura K, Okumura K, Zineldeen DH, Shimada M, Niida H, Mizuno T, Hanaoka F, Nakanishi M (2009) Cyclin A-Cdk1 regulates the origin firing program in mammalian cells. Proc Natl Acad Sci U S A 106: 3184–3189. 10.1073/pnas.0809350106 PubMed DOI PMC

Kim Y, Starostina NG, Kipreos ET (2008) The CRL4Cdt2 ubiquitin ligase targets the degradation of p21Cip1 to control replication licensing. Genes Dev 22: 2507–2519. 10.1101/gad.1703708 PubMed DOI PMC

Koundrioukoff S, Jónsson ZO, Hasan S, de Jong RN, van der Vliet PC, Hottiger MO, Hübscher U (2000) A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation. J Biol Chem 275: 22882–22887. 10.1074/jbc.M001850200 PubMed DOI

Krenning L, Feringa FM, Shaltiel IA, van den Berg J, Medema RH (2014) Transient activation of p53 in G2 phase is sufficient to induce senescence. Mol Cell 55: 59–72. 10.1016/j.molcel.2014.05.007 PubMed DOI

Laoukili J, Alvarez M, Meijer LA, Stahl M, Mohammed S, Kleij L, Heck AJ, Medema RH (2008) Activation of FoxM1 during G2 requires cyclin A/Cdk-dependent relief of autorepression by the FoxM1 N-terminal domain. Mol Cell Biol 28: 3076–3087. 10.1128/MCB.01710-07 PubMed DOI PMC

Lemmens B, Hegarat N, Akopyan K, Sala-Gaston J, Bartek J, Hochegger H, Lindqvist A (2018) DNA replication determines timing of mitosis by restricting CDK1 and PLK1 activation. Mol Cell 71: 117–128.e3. 10.1016/j.molcel.2018.05.026 PubMed DOI PMC

Lemmens B, Lindqvist A (2019) DNA replication and mitotic entry: A brake model for cell cycle progression. J Cell Biol 218: 3892–3902. 10.1083/jcb.201909032 PubMed DOI PMC

Li J-A, Liu BC, Song Y, Chen X (2018) Cyclin A2 regulates symmetrical mitotic spindle formation and centrosome amplification in human colon cancer cells. Am J Transl Res 10: 2669–2676. PubMed PMC

Lindqvist A, Rodriguez-Bravo V, Medema RH (2009) The decision to enter mitosis: Feedback and redundancy in the mitotic entry network. J Cell Biol 185: 193–202. 10.1083/jcb.200812045 PubMed DOI PMC

Lukas C, Sorensen CS, Kramer E, Santoni-Rugiu E, Lindeneg C, Peters JM, Bartek J, Lukas J (1999) Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex. Nature 401: 815–818. 10.1038/44611 PubMed DOI

Macleod KF, Sherry N, Hannon G, Beach D, Tokino T, Kinzler K, Vogelstein B, Jacks T (1995) p53-dependent and independent expression of p21 during cell growth, differentiation, and DNA damage. Genes Dev 9: 935–944. 10.1101/gad.9.8.935 PubMed DOI

Macurek L, Lindqvist A, Lim D, Lampson MA, Klompmaker R, Freire R, Clouin C, Taylor SS, Yaffe MB, Medema RH (2008) Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery. Nature 455: 119–123. 10.1038/nature07185 PubMed DOI

Maridor G, Gallant P, Golsteyn R, Nigg EA (1993) Nuclear localization of vertebrate cyclin A correlates with its ability to form complexes with cdk catalytic subunits. J Cell Sci 106: 535–544. https://jcs.biologists.org/content/106/2/535 PubMed

Merrick KA, Larochelle S, Zhang C, Allen JJ, Shokat KM, Fisher RP (2008) Distinct activation pathways confer cyclin-binding specificity on Cdk1 and Cdk2 in human cells. Mol Cell 32: 662–672. 10.1016/j.molcel.2008.10.022 PubMed DOI PMC

Mitra J, Enders GH (2004) Cyclin A/Cdk2 complexes regulate activation of Cdk1 and Cdc25 phosphatases in human cells. Oncogene 23: 3361–3367. 10.1038/sj.onc.1207446 PubMed DOI PMC

Moore JD, Yang J, Truant R, Kornbluth S (1999) Nuclear import of Cdk/cyclin complexes: Identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1. J Cell Biol 144: 213–224. 10.1083/jcb.144.2.213 PubMed DOI PMC

Müllers E, Cascales HS, Burdova K, Macurek L, Lindqvist A (2017) Residual Cdk1/2 activity after DNA damage promotes senescence. Aging Cell 16: 575–584. 10.1111/acel.12588 PubMed DOI PMC

Müllers E, Silva Cascales H, Jaiswal H, Saurin AT, Lindqvist A (2014) Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase. Cell Cycle 13: 2733–2743. 10.4161/15384101.2015.945831 PubMed DOI PMC

Nishitani H, Shiomi Y, Iida H, Michishita M, Takami T, Tsurimoto T (2008) CDK inhibitor p21 is degraded by a proliferating cell nuclear antigen-coupled Cul4-DDB1Cdt2 pathway during S phase and after UV irradiation. J Biol Chem 283: 29045–29052. 10.1074/jbc.M806045200 PubMed DOI PMC

Oakes V, Wang W, Harrington B, Lee WJ, Beamish H, Chia KM, Pinder A, Goto H, Inagaki M, Pavey S, et al. (2014) Cyclin A/Cdk2 regulates Cdh1 and claspin during late S/G2 phase of the cell cycle. Cell Cycle 13: 3302–3311. 10.4161/15384101.2014.949111 PubMed DOI PMC

Örd M, Loog M (2019) How the cell cycle clock ticks. Mol Biol Cell 30: 169–172. 10.1091/mbc.E18-05-0272 PubMed DOI PMC

Pagano M, Pepperkok R, Verde F, Ansorge W, Draetta G (1992) Cyclin A is required at two points in the human cell cycle. EMBO J 11: 961–971. 10.1002/j.1460-2075.1992.tb05135.x PubMed DOI PMC

Parrilla A, Cirillo L, Thomas Y, Gotta M, Pintard L, Santamaria A (2016) Mitotic entry: The interplay between Cdk1, Plk1 and Bora. Cell Cycle 15: 3177–3182. 10.1080/15384101.2016.1249544 PubMed DOI PMC

Pechackova S, Burdova K, Benada J, Kleiblova P, Jenikova G, Macurek L (2016) Inhibition of WIP1 phosphatase sensitizes breast cancer cells to genotoxic stress and to MDM2 antagonist nutlin-3. Oncotarget 7: 14458–14475. 10.18632/oncotarget.7363 PubMed DOI PMC

Petersen BO, Lukas J, Sorensen CS, Bartek J, Helin K (1999) Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization. EMBO J 18: 396–410. 10.1093/emboj/18.2.396 PubMed DOI PMC

Pines J, Hunter T (1991) Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport. J Cell Biol 115: 1–17. 10.1083/jcb.115.1.1 PubMed DOI PMC

Pines J, Hunter T (1994) The differential localization of human cyclins A and B is due to a cytoplasmic retention signal in cyclin B. EMBO J 13: 3772–3781. 10.1002/j.1460-2075.1994.tb06688.x PubMed DOI PMC

Qi F, Chen Q, Chen H, Yan H, Chen B, Xiang X, Liang C, Yi Q, Zhang M, Cheng H, et al. (2018) WAC promotes polo-like kinase 1 activation for timely mitotic entry. Cell Rep 24: 546–556. 10.1016/j.celrep.2018.06.087 PubMed DOI

Rodríguez-Vilarrupla A, Díaz C, Canela N, Rahn HP, Bachs O, Agell N (2002) Identification of the nuclear localization signal of p21(cip1) and consequences of its mutation on cell proliferation. FEBS Lett 531: 319–323. 10.1016/s0014-5793(02)03549-4 PubMed DOI

Saldivar JC, Hamperl S, Bocek MJ, Chung M, Bass TE, Cisneros-Soberanis F, Samejima K, Xie L, Paulson JR, Earnshaw WC, et al. (2018) An intrinsic S/G2 checkpoint enforced by ATR. Science 361: 806–810. 10.1126/science.aap9346 PubMed DOI PMC

Satyanarayana A, Kaldis P (2009) Mammalian cell-cycle regulation: Several Cdks, numerous cyclins and diverse compensatory mechanisms. Oncogene 28: 2925–2939. 10.1038/onc.2009.170 PubMed DOI

Seki A, Coppinger JA, Jang CY, Yates JR, Fang G (2008) Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry. Science 320: 1655–1658. 10.1126/science.1157425 PubMed DOI PMC

Sheng C, Mendler IH, Rieke S, Snyder P, Jentsch M, Friedrich D, Drossel B, Loewer A (2019) PCNA-mediated degradation of p21 coordinates the DNA damage response and cell cycle regulation in individual cells. Cell Rep 27: 48–58.e7. 10.1016/j.celrep.2019.03.031 PubMed DOI

Sørensen CS, Syljuåsen RG (2012) Safeguarding genome integrity: The checkpoint kinases ATR, CHK1 and WEE1 restrain CDK activity during normal DNA replication. Nucleic Acids Res 40: 477–486. 10.1093/nar/gkr697 PubMed DOI PMC

Swaffer MP, Jones AW, Flynn HR, Snijders AP, Nurse P (2016) CDK substrate phosphorylation and ordering the cell cycle. Cell 167: 1750–1761.e16. 10.1016/j.cell.2016.11.034 PubMed DOI PMC

Takizawa CG, Morgan DO (2000) Control of mitosis by changes in the subcellular location of cyclin-B1-Cdk1 and Cdc25C. Curr Opin Cell Biol 12: 658–665. 10.1016/s0955-0674(00)00149-6 PubMed DOI

Taniguchi E, Toyoshima-Morimoto F, Nishida E (2002) Nuclear translocation of plk1 mediated by its bipartite nuclear localization signal. J Biol Chem 277: 48884–48888. 10.1074/jbc.M206307200 PubMed DOI

Tavernier N, Noatynska A, Panbianco C, Martino L, Van Hove L, Schwager F, Léger T, Gotta M, Pintard L (2015) Cdk1 phosphorylates SPAT-1/Bora to trigger PLK-1 activation and drive mitotic entry in C. elegans embryos. J Cell Biol 208: 661–669. 10.1083/jcb.201408064 PubMed DOI PMC

Thomas Y, Cirillo L, Panbianco C, Martino L, Tavernier N, Schwager F, Van Hove L, Joly N, Santamaria A, Pintard L, et al. (2016) Cdk1 phosphorylates SPAT-1/Bora to promote Plk1 activation in C. elegans and human cells. Cell Rep 15: 510–518. 10.1016/j.celrep.2016.03.049 PubMed DOI

Thul PJ, Åkesson L, Wiking M, Mahdessian D, Geladaki A, Blal HA, Alm T, Asplund A, Björk L, Breckels LM, et al. (2017) A subcellular map of the human proteome. Science 356: eaal3321 10.1126/science.aal3321 PubMed DOI

Trembecka-Lucas DO, Trembecka-Lucas DO, Szczurek AT, Szczurek AT, Dobrucki JW, Dobrucki JW (2013) Dynamics of the HP1β-PCNA-containing complexes in DNA replication and repair. Nucleus 4: 74–82. 10.4161/nucl.23683 PubMed DOI PMC

Tsang WY, Wang L, Chen Z, Sanchez I, Dynlacht BD (2007) SCAPER, a novel cyclin A-interacting protein that regulates cell cycle progression. J Cell Biol 178: 621–633. 10.1083/jcb.200701166 PubMed DOI PMC

Uetake Y, Sluder G (2010) Prolonged prometaphase blocks daughter cell proliferation despite normal completion of mitosis. Curr Biol 20: 1666–1671. 10.1016/j.cub.2010.08.018 PubMed DOI PMC

Vigneron S, Sundermann L, Labbé JC, Pintard L, Radulescu O, Castro A, Lorca T (2018) Cyclin A-cdk1-dependent phosphorylation of Bora is the triggering factor promoting mitotic entry. Dev Cell 45: 637–650.e7. 10.1016/j.devcel.2018.05.005 PubMed DOI

Woodman PG, Adamczewski JP, Hunt T, Warren G (1993) In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase. Mol Biol Cell 4: 541–553. 10.1091/mbc.4.5.541 PubMed DOI PMC

Zerjatke T, Gak IA, Kirova D, Fuhrmann M, Daniel K, Gonciarz M, Müller D, Glauche I, Mansfeld J (2017) Quantitative cell cycle analysis based on an endogenous all-in-one reporter for cell tracking and classification. Cell Rep 19: 1953–1966. 10.1016/j.celrep.2017.05.022 PubMed DOI PMC