Cyclin A/Cdk1 modulates Plk1 activity in prometaphase to regulate kinetochore-microtubule attachment stability.

Ana Maria G Dumitru,Scott F Rusin,Amber E M Clark,Arminja N Kettenbach,Duane A Compton

doi:10.7554/elife.29303

Abstract

The fidelity of chromosome segregation in mitosis is safeguarded by the precise regulation of kinetochore microtubule (k-MT) attachment stability. Previously, we demonstrated that Cyclin A/Cdk1 destabilizes k-MT attachments to promote faithful chromosome segregation. Here, we use quantitative phosphoproteomics to identify 156 Cyclin A/Cdk1 substrates in prometaphase. One Cyclin A/Cdk1 substrate is myosin phosphatase targeting subunit 1 (MYPT1), and we show that MYPT1 localization to kinetochores depends on Cyclin A/Cdk1 activity and that MYPT1 destabilizes k-MT attachments by negatively regulating Plk1 at kinetochores. Thus, Cyclin A/Cdk1 phosphorylation primes MYPT1 for Plk1 binding. Interestingly, priming of PBIP1 by Plk1 itself (self-priming) increased in MYPT1-depleted cells showing that MYPT1 provides a molecular link between the processes of Cdk1-dependent priming and self-priming of Plk1 substrates. These data demonstrate cross-regulation between Cyclin A/Cdk1-dependent and Plk1-dependent phosphorylation of substrates during mitosis to ensure efficient correction of k-MT attachment errors necessary for high mitotic fidelity.

Highlights

Mitotic cells rely on the precise regulation of the stability of microtubule attachments to kinetochores (k-MT attachments) on chromosomes to ensure genetic fidelity during cell division (Bakhoum et al, 2009a; Bakhoum et al, 2009b; Walczak et al, 2010)
To identify Cyclin A/Cdk1-specific mitotic targets in prometaphase, we utilized Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) (Ong et al, 2002) to conduct a bioinformatics-assisted phosphoproteomic screen comparing phosphorylation of protein peptides from cells arrested in mitosis with high versus low levels of Cyclin A
The capacity of kinetochores to release their attached microtubules is essential for faithful chromosome segregation (Nicklas and Ward, 1994), and we and others have previously established a mitotic role for Cyclin A/Cdk1 in destabilizing kinetochore microtubule (k-MT) attachments to promote efficient correction of k-MT attachment errors (Kabeche and Compton, 2013; Zhang et al, 2017)

Summary

Introduction

Mitotic cells rely on the precise regulation of the stability of microtubule attachments to kinetochores (k-MT attachments) on chromosomes to ensure genetic fidelity during cell division (Bakhoum et al, 2009a; Bakhoum et al, 2009b; Walczak et al, 2010). Kinetochore-MT attachments must be sufficiently stable to achieve microtubule occupancy at kinetochores to move chromosomes and to satisfy the spindle assembly checkpoint (SAC) in a timely manner (Bakhoum et al, 2009b; Bakhoum and Compton, 2012; Musacchio and Salmon, 2007; McEwen and Dong, 2009). Microtubules must rapidly detach from kinetochores to promote efficient correction of erroneously oriented k-MT attachments (Thompson et al, 2010; Maiato et al, 2004). Destabilizing k-MT attachments was shown to be sufficient to restore faithful chromosome segregation to tumor cells that otherwise exhibited exceptionally high chromosome mis-segregation rates (Bakhoum et al, 2009b; Kabeche and Compton, 2013; Stolz et al, 2010). There is a causal relationship between the stability of k-MT attachments and the propensity for chromosome mis-segregation

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Conclusion