title: Requirement for PLK1 kinase activity in the maintenance of a robust spindle assembly checkpoint.
abstract: During mitotic arrest induced by microtubule targeting drugs, the weakening of the spindle assembly checkpoint (SAC) allows cells to progress through the cell cycle without chromosome segregation occurring. PLK1 kinase plays a major role in mitosis and emerging evidence indicates that PLK1 is also involved in establishing the checkpoint and maintaining SAC signalling. However, mechanistically, the role of PLK1 in the SAC is not fully understood, with several recent reports indicating that it can cooperate with either one of the major checkpoint kinases, Aurora B or MPS1. In this study, we assess the role of PLK1 in SAC maintenance. We find that in nocodazole-arrested U2OS cells, PLK1 activity is continuously required for maintaining Aurora B protein localisation and activity at kinetochores. Consistent with published data we find that upon PLK1 inhibition, phosphoThr3-H3, a marker of Haspin activity, is reduced. Intriguingly, Aurora B inhibition causes PLK1 to relocalise from kinetochores into fewer and much larger foci, possibly due to incomplete recruitment of outer kinetochore proteins. Importantly, PLK1 inhibition, together with partial inhibition of Aurora B, allows efficient SAC override to occur. This phenotype is more pronounced than the phenotype observed by combining the same PLK1 inhibitors with partial MPS1 inhibition. We also find that PLK1 inhibition does not obviously cooperate with Haspin inhibition to promote SAC override. These results indicate that PLK1 is directly involved in maintaining efficient SAC signalling, possibly by cooperating in a positive feedback loop with Aurora B, and that partially redundant mechanisms exist which reinforce the SAC.
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Aisling
authors.initials: A
authors.lastname: O'Connor
authors.affiliation: Max-Planck Institute of Molecular Physiology, Department of Mechanistic Cell Biology, Dortmund 44227, Germany.
authors.forename: Stefano
authors.initials: S
authors.lastname: Maffini
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Rainey
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Kaczmarczyk
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland.
authors.forename: David
authors.initials: D
authors.lastname: Gaboriau
authors.affiliation: Max-Planck Institute of Molecular Physiology, Department of Mechanistic Cell Biology, Dortmund 44227, Germany Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstrasse, Essen 45141, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Musacchio
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Galway, Ireland Corrado.Santocanale@nuigalway.ie.
authors.forename: Corrado
authors.initials: C
authors.lastname: Santocanale
keywords: Aurora B; Cell-Cycle; Kinase inhibitors; MPS1, Haspin, Spindle assembly checkpoint; PLK1