Abstract
Plk1 (polo-like kinase 1) is an evolutionarily conserved serine/threonine kinase instrumental for mitotic entry and progression. Beyond these canonical functions, Plk1 also regulates cell polarization and cell fate during asymmetric cell divisions in C. elegans and D. melanogaster. Plk1 contains a specialized phosphoserine–threonine binding domain, the polo-box domain (PBD), which localizes and concentrates the kinase at its various sites of action within the cell in space and time. Here we present protocols to express and purify the C. elegans Plk1 kinase along with biochemical and phosphoproteomic approaches to interrogate the PBD interactome and to dissect Plk1 substrate interactions. These protocols are most suitable for the identification of Plk1 targets in C. elegans embryos but can be easily adapted to identify and study Plk1 substrates from any source.”
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References
Archambault V, Glover DM (2009) Polo-like kinases: conservation and divergence in their functions and regulation. Nat Rev Mol Cell Biol 10:265–275
Zitouni S, Nabais C, Jana SC et al (2014) Polo-like kinases: structural variations lead to multiple functions. Nat Rev Mol Cell Biol 15:433–452
Combes G, Alharbi I, Braga LG, Elowe S (2017) Playing polo during mitosis: PLK1 takes the lead. Oncogene 36:4819–4827
Pintard L, Archambault V (2018) A unified view of spatio-temporal control of mitotic entry: polo kinase as the key. Open Biol 8:180114. https://doi.org/10.1098/rsob.180114
Cheng KY, Lowe ED, Sinclair J et al (2003) The crystal structure of the human polo-like kinase-1 polo box domain and its phospho-peptide complex. EMBO J 22:5757–5768
Elia AE, Rellos P, Haire LF et al (2003) The molecular basis for phosphodependent substrate targeting and regulation of Plks by the polo-box domain. Cell 115:83–95
Elia AE, Cantley LC, Yaffe MB (2003) Proteomic screen finds pSer/pThr-binding domain localizing Plk1 to mitotic substrates. Science 299:1228–1231
Nigg EA (2001) Mitotic kinases as regulators of cell division and its checkpoints. Nat Rev Mol Cell Biol 2:21–32
Elowe S, Hümmer S, Uldschmid A et al (2007) Tension-sensitive Plk1 phosphorylation on BubR1 regulates the stability of kinetochore microtubule interactions. Genes Dev 21:2205–2219
Qi W, Tang Z, Yu H (2006) Phosphorylation- and polo-box-dependent binding of Plk1 to Bub1 is required for the kinetochore localization of Plk1. Mol Biol Cell 17:3705–3716
Chan EH, Santamaria A, Sillje HH, Nigg EA (2008) Plk1 regulates mitotic Aurora A function through betaTrCP-dependent degradation of hBora. Chromosoma 117:457–469
Nakajima H, Toyoshima-Morimoto F, Taniguchi E, Nishida E (2003) Identification of a consensus motif for Plk (Polo-like kinase) phosphorylation reveals Myt1 as a Plk1 substrate. J Biol Chem 278:25277–25280
Kettenbach AN, Schweppe DK, Faherty BK et al (2011) Quantitative phosphoproteomics identifies substrates and functional modules of Aurora and Polo-like kinase activities in mitotic cells. Sci Signal 4:rs5
Neef R, Preisinger C, Sutcliffe J et al (2003) Phosphorylation of mitotic kinesin-like protein 2 by polo-like kinase 1 is required for cytokinesis. J Cell Biol 162:863–875
Kang YH, Park JE, Yu LR et al (2006) Self-regulated Plk1 recruitment to kinetochores by the Plk1-PBIP1 interaction is critical for proper chromosome segregation. Mol Cell 24:409–422
Park JE, Soung NK, Johmura Y et al (2010) Polo-box domain: a versatile mediator of polo-like kinase function. Cell Mol Life Sci 67:1957–1970
Saurin AT (2018) Kinase and phosphatase cross-talk at the kinetochore. Front Cell Dev Biol 6:62
Noatynska A, Tavernier N, Gotta M, Pintard L (2013) Coordinating cell polarity and cell cycle progression: what can we learn from flies and worms? Open Biol 3:130083
Kim AJ, Griffin EE (2020) PLK-1 regulation of asymmetric cell division in the Early C. elegans embryo. Front cell. Dev Biol 8:632253
Taylor SJP, Bel Borja L, Soubigou F et al (2023) BUB-1 and CENP-C recruit PLK-1 to control chromosome alignment and segregation during meiosis I in C. elegans oocytes. Elife 12:e84057
Endicott JA, Noble ME, Johnson LN (2012) The structural basis for control of eukaryotic protein kinases. Annu Rev Biochem 81:587–613
Macurek L, Lindqvist A, Lim D et al (2008) Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery. Nature 455:119–123
Tavernier N, Noatynska A, Panbianco C et al (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
Lowery DM, Clauser KR, Hjerrild M et al (2007) Proteomic screen defines the polo-box domain interactome and identifies Rock2 as a Plk1 substrate. EMBO J 26:2262–2273
Nkombo Nkoula S, Velez-Aguilera G, Ossareh-Nazari B, Van Hove L, Ayuso C, Legros V, Chevreux G, Thomas L, Seydoux G, Askjaer P, Pintard L (2023) Mechanisms of nuclear pore complex disassembly by the mitotic Polo-like kinase 1 (PLK-1) in C. elegans embryos. Sci Adv 9:eadf7826
Stiernagle T (2006) Maintenance of C. elegans. WormBook 1–11
Singh P, Pesenti ME, Maffini SC, et al (2021) BUB1 and CENP-U, primed by CDK1, are the main PLK1 kinetochore receptors in mitosis. Mol Cell 81:67–87.e9
Wu Y, Li Q, Chen XZ (2007) Detecting protein-protein interactions by far western blotting. Nat Protoc 2:3278–3284
Martino L, Morchoisne-Bolhy S, Cheerambathur DK et al (2017) Channel nucleoporins recruit PLK-1 to nuclear pore complexes to direct nuclear envelope breakdown in C. elegans. Dev Cell 43:157–171.e7
Velez-Aguilera G, Nkombo Nkoula S, Ossareh-Nazari B et al (2020) PLK-1 promotes the merger of the parental genome into a single nucleus by triggering lamina disassembly. elife 9:e59510
Brooks KK, Liang B, Watts JL (2009) The influence of bacterial diet on fat storage in C. elegans. PLoS One 4:e7545
Acknowledgments
We thank P. Moussounda for media preparation and Véronique Legros and Guillaume Chevreux, from the Proteomic platform, for phosphopeptide purification and tandem mass spectrometry analysis. We thank R. Karess for critical reading of the manuscript. We thank all members of the team for stimulating discussions. Work in LP laboratory is supported by grants from Agence Nationale pour la Recherche (ANR), France, ANR-22-CE13-0022 (LP), La Ligue Nationale Contre le Cancer, Equipe Labellisée, France, and Idex Université Paris Cité, ANR-18-IDEX-0001. Griselda Velez-Aguilera is supported by a postdoctoral fellowship from “Secretaría de Educación, Ciencia, Tecnología e Innovación de la Ciudad de México,” CMSECTEI/201/2022.
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Velez-Aguilera, G., Ossareh-Nazari, B., Pintard, L. (2024). Dissecting the Multiple Functions of the Polo-Like Kinase 1 in the C. elegans Zygote. In: Castro, A., Lacroix, B. (eds) Cell Cycle Control. Methods in Molecular Biology, vol 2740. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3557-5_4
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DOI: https://doi.org/10.1007/978-1-0716-3557-5_4
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