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FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery

Cells recovering from the G2/M DNA damage checkpoint rely more on Aurora A-PLK1 signaling than cells progressing through an unperturbed G2 phase, but the reason for this discrepancy is not known. Here, we devised a method based on a FRET reporter for PLK1 activity to sort cells in distinct populatio...

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Detalles Bibliográficos
Autores principales: Lafranchi, Lorenzo, Müllers, Erik, Rutishauser, Dorothea, Lindqvist, Arne
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564076/
https://www.ncbi.nlm.nih.gov/pubmed/32961751
http://dx.doi.org/10.3390/cells9092126
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author Lafranchi, Lorenzo
Müllers, Erik
Rutishauser, Dorothea
Lindqvist, Arne
author_facet Lafranchi, Lorenzo
Müllers, Erik
Rutishauser, Dorothea
Lindqvist, Arne
author_sort Lafranchi, Lorenzo
collection PubMed
description Cells recovering from the G2/M DNA damage checkpoint rely more on Aurora A-PLK1 signaling than cells progressing through an unperturbed G2 phase, but the reason for this discrepancy is not known. Here, we devised a method based on a FRET reporter for PLK1 activity to sort cells in distinct populations within G2 phase. We employed mass spectroscopy to characterize changes in protein levels through an unperturbed G2 phase and validated that ATAD2 levels decrease in a proteasome-dependent manner. Comparing unperturbed cells with cells recovering from DNA damage, we note that at similar PLK1 activities, recovering cells contain higher levels of Cyclin B1 and increased phosphorylation of CDK1 targets. The increased Cyclin B1 levels are due to continuous Cyclin B1 production during a DNA damage response and are sustained until mitosis. Whereas partial inhibition of PLK1 suppresses mitotic entry more efficiently when cells recover from a checkpoint, partial inhibition of CDK1 suppresses mitotic entry more efficiently in unperturbed cells. Our findings provide a resource for proteome changes during G2 phase, show that the mitotic entry network is rewired during a DNA damage response, and suggest that the bottleneck for mitotic entry shifts from CDK1 to PLK1 after DNA damage.
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spelling pubmed-75640762020-10-27 FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery Lafranchi, Lorenzo Müllers, Erik Rutishauser, Dorothea Lindqvist, Arne Cells Article Cells recovering from the G2/M DNA damage checkpoint rely more on Aurora A-PLK1 signaling than cells progressing through an unperturbed G2 phase, but the reason for this discrepancy is not known. Here, we devised a method based on a FRET reporter for PLK1 activity to sort cells in distinct populations within G2 phase. We employed mass spectroscopy to characterize changes in protein levels through an unperturbed G2 phase and validated that ATAD2 levels decrease in a proteasome-dependent manner. Comparing unperturbed cells with cells recovering from DNA damage, we note that at similar PLK1 activities, recovering cells contain higher levels of Cyclin B1 and increased phosphorylation of CDK1 targets. The increased Cyclin B1 levels are due to continuous Cyclin B1 production during a DNA damage response and are sustained until mitosis. Whereas partial inhibition of PLK1 suppresses mitotic entry more efficiently when cells recover from a checkpoint, partial inhibition of CDK1 suppresses mitotic entry more efficiently in unperturbed cells. Our findings provide a resource for proteome changes during G2 phase, show that the mitotic entry network is rewired during a DNA damage response, and suggest that the bottleneck for mitotic entry shifts from CDK1 to PLK1 after DNA damage. MDPI 2020-09-19 /pmc/articles/PMC7564076/ /pubmed/32961751 http://dx.doi.org/10.3390/cells9092126 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Lafranchi, Lorenzo
Müllers, Erik
Rutishauser, Dorothea
Lindqvist, Arne
FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery
title FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery
title_full FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery
title_fullStr FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery
title_full_unstemmed FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery
title_short FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery
title_sort fret-based sorting of live cells reveals shifted balance between plk1 and cdk1 activities during checkpoint recovery
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564076/
https://www.ncbi.nlm.nih.gov/pubmed/32961751
http://dx.doi.org/10.3390/cells9092126
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