Inhibition of Polo-like kinase 1 during the DNA damage response is mediated through loss of Aurora A recruitment by Bora

Abstract

When cells in G2 phase are challenged with DNA damage, several key mitotic regulators such as Cdk1/Cyclin B, Aurora A and Plk1 are inhibited to prevent entry into mitosis. Here we have studied how inhibition of Plk1 is established after DNA damage. Using a Förster resonance energy transfer (FRET)-based biosensor for Plk1 activity, we show that inhibition of Plk1 after DNA damage occurs with relatively slow kinetics and is entirely dependent on loss of Plk1-T210 phosphorylation. As T210 is phosphorylated by the kinase Aurora A in conjunction with its co-factor Bora, we investigated how they are affected by DNA damage. Interestingly, we find that the interaction between Bora and Plk1 remains intact during the early phases of the DNA damage response (DDR), whereas Plk1 activity is already inhibited at this stage. Expression of an Aurora A mutant that is refractory to inhibition by the DDR failed to prevent inhibition of Plk1 and loss of T210 phosphorylation, suggesting that inhibition of Plk1 may be established by perturbing recruitment of Aurora A by Bora. Indeed, expression of a fusion in which Aurora A was directly coupled to Bora prevented DNA damage-induced inhibition of Plk1 activity, as well as inhibition of T210 phosphorylation. Taken together, these data demonstrate that DNA damage affects the function of Aurora A at multiple levels: both by direct inhibition of Aurora A activity, as well as by perturbing the interaction with its co-activator Bora. We propose that the DDR targets recruitment of Aurora A to the Plk1/Bora complex to prevent activation of Plk1 during DNA damage in G2.

DNA damage kinase signaling: checkpoint and repair at 30 years

“…A more in-depth review exploring Cdc5's complex relationship with the DNA damage response in yeast can be found in Botchkarev and Haber (2018). Similar to its budding yeast counterpart, the mammalian polo-like kinase PLK1 is also inhibited by DNA damage signaling, albeit in an indirect manner (Fig 3;Bruinsma et al, 2017;Qin et al, 2013).…”

Regulating a key mitotic regulator, polo‐like kinase 1 (PLK1)

“…The binding of PLK1 to its scaffold can assist in regulating PLK1’s activity in three possible ways: (1) To augment or enhance its ability to phosphorylate downstream substrates; (2) To insulate or sequester it to a specific locale; (3) To terminate or impede its ability to phosphorylate downstream substrates (Langeberg & Scott, ). While there is much known about PLK1 and the mitotic scaffold Bora (Bruinsma et al, ; Macůrek et al, ; Seki et al, ; Thomas et al, ), here, we will focus on what is known about PLK1 scaffolds specifically at mitotic centrosomes and our recent study involving PLK1 and its PCM localized scaffold Gravin (Figure ). Strikingly, the two mitotic centrosomes, which appear to be symmetric in nature, are actually asymmetric structures (Nigg & Stearns, ).…”

“…The development of a PLK1‐FRET biosensor allowed, for the first time, the activity of PLK1 to be examined in an in vivo setting throughout the cell cycle (Bruinsma et al, ; Macůrek et al, ). By manipulating a previously developed Aurora B kinase activity biosensor (Fuller et al, ), a biosensor was developed that possessed a donor (CFP) and acceptor (YFP), a phospho‐binding domain (FHA2, Forkhead‐Associated Domain 2), and a PLK1 specific phosphorylation sequence (Myt1).…”

ATM /Wip1 activities at chromatin control Plk1 re‐activation to determine G2 checkpoint duration

“…Key targets of Chk1/Chk2/MK2 are Cdc25 phosphatases and their degradation or functional inactivation results in a rapid decrease in Cdk1 activity and suppression of cell cycle progression (Peng et al, 1997;Mailand et al, 2000;Reinhardt et al, 2007). In parallel, Plk1 is inactivated by ATM-mediated dephosphorylation of Thr210 by phosphatase PP2A/B55a and by ATR-mediated degradation of Bora, the co-factor necessary for activation of Plk1 (Qin et al, 2013;Wang et al, 2015;Bruinsma et al, 2017). In addition to this rapid response, a checkpoint is reinforced by activation of p53 and expression of the Cdk inhibitor p21 cip/waf and also by premature activation of APC/C-Cdh1 that results in degradation of multiple proteins including Plk1 (Bunz et al, 1998;de Boer et al, 2016).…”