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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., (© 2021 Silva Cascales et al.)

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.

To maintain genome stability, cells need to replicate their DNA before dividing. Upon completion of bulk DNA synthesis, the mitotic kinases CDK1 and PLK1 become active and drive entry into mitosis. Here, we have tested the hypothesis that DNA replication determines the timing of mitotic kinase activation. Using an optimized double-degron system, together with kinase inhibitors to enforce tight inhibition of key proteins, we find that human cells unable to initiate DNA replication prematurely enter mitosis. Preventing DNA replication licensing and/or firing causes prompt activation of CDK1 and PLK1 in S phase. In the presence of DNA replication, inhibition of CHK1 and p38 leads to premature activation of mitotic kinases, which induces severe replication stress. Our results demonstrate that, rather than merely a cell cycle output, DNA replication is an integral signaling component that restricts activation of mitotic kinases. DNA replication thus functions as a brake that determines cell cycle duration., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

After DNA damage, the cell cycle is arrested to avoid propagation of mutations. Arrest in G2 phase is initiated by ATM-/ATR-dependent signaling that inhibits mitosis-promoting kinases such as Plk1. At the same time, Plk1 can counteract ATR-dependent signaling and is required for eventual resumption of the cell cycle. However, what determines when Plk1 activity can resume remains unclear. Here, we use FRET-based reporters to show that a global spread of ATM activity on chromatin and phosphorylation of ATM targets including KAP1 control Plk1 re-activation. These phosphorylations are rapidly counteracted by the chromatin-bound phosphatase Wip1, allowing cell cycle restart despite persistent ATM activity present at DNA lesions. Combining experimental data and mathematical modeling, we propose a model for how the minimal duration of cell cycle arrest is controlled. Our model shows how cell cycle restart can occur before completion of DNA repair and suggests a mechanism for checkpoint adaptation in human cells., (© 2017 The Authors.)

Polo-like kinase-1 (Plk1) is required for proper cell division. Activation of Plk1 requires phosphorylation on a conserved threonine in the T-loop of the kinase domain (T210). Plk1 is first phosphorylated on T210 in G2 phase by the kinase Aurora-A, in concert with its cofactor Bora. However, Bora was shown to be degraded prior to entry into mitosis, and it is currently unclear how Plk1 activity is sustained in mitosis. Here we show that the Bora-Aurora-A complex remains the major activator of Plk1 in mitosis. We show that a small amount of Aurora-A activity is sufficient to phosphorylate and activate Plk1 in mitosis. In addition, a fraction of Bora is retained in mitosis, which is essential for continued Aurora-A-dependent T210 phosphorylation of Plk1. We find that once Plk1 is activated, minimal amounts of the Bora-Aurora-A complex are sufficient to sustain Plk1 activity. Thus, the activation of Plk1 by Aurora-A may function as a bistable switch; highly sensitive to inhibition of Aurora-A in its initial activation, but refractory to fluctuations in Aurora-A activity once Plk1 is fully activated. This provides a cell with robust Plk1 activity once it has committed to mitosis.

Cdk1 and Plk1/Plx1 activation leads to their inactivation through negative feedback loops. Cdk1 deactivates itself by activating the APC/C, consequently generating embryonic cell cycle oscillations. APC/C inhibition by the mitotic checkpoint in somatic cells and the cytostatic factor (CSF) in oocytes sustain the mitotic state. Plk1/Plx1 targets its co-activator Bora for degradation, but it remains unclear how embryonic oscillations in Plx1 activity are generated, and how Plk1/Plx1 activity is sustained during mitosis. We show that Plx1-mediated degradation of Bora in interphase generates oscillations in Plx1 activity and is essential for development. In CSF extracts, phosphorylation of Bora on the Cdk consensus site T52 blocks Bora degradation. Upon fertilization, Calcineurin dephosphorylates T52, triggering Plx1 oscillations. Similarly, we find that GFP-Bora is degraded when Plk1 activity spreads to somatic cell cytoplasm before mitosis. Interestingly, GFP-Bora degradation stops upon mitotic entry when Cdk1 activity is high. We hypothesize that Cdk1 controls Bora through an incoherent feedforward loop synchronizing the activities of mitotic kinases.

Overactivation of both Polo-like kinase-1 (Plk1) and Aurora-A is linked to cancer development, and small-molecule inhibitors that target these kinases are currently tested as anticancer drugs. Here, we discuss recent advances in the understanding of the functional crosstalk between Plk1 and Aurora-A before and during mitosis. Several recent findings have led to a better appreciation of how the activities of these distinct mitotic kinases are intertwined. Such insight is important for the expected utility of small-molecule inhibitors targeting Plk1 or Aurora-A, and it might help us to improve their application.

Polo-like kinase-1 (PLK1) is an essential mitotic kinase regulating multiple aspects of the cell division process. Activation of PLK1 requires phosphorylation of a conserved threonine residue (Thr 210) in the T-loop of the PLK1 kinase domain, but the kinase responsible for this has not yet been affirmatively identified. Here we show that in human cells PLK1 activation occurs several hours before entry into mitosis, and requires aurora A (AURKA, also known as STK6)-dependent phosphorylation of Thr 210. We find that aurora A can directly phosphorylate PLK1 on Thr 210, and that activity of aurora A towards PLK1 is greatly enhanced by Bora (also known as C13orf34 and FLJ22624), a known cofactor for aurora A (ref. 7). We show that Bora/aurora-A-dependent phosphorylation is a prerequisite for PLK1 to promote mitotic entry after a checkpoint-dependent arrest. Importantly, expression of a PLK1-T210D phospho-mimicking mutant partially overcomes the requirement for aurora A in checkpoint recovery. Taken together, these data demonstrate that the initial activation of PLK1 is a primary function of aurora A.