Abstract
Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G(2)/M DNA damage checkpoint and efficient checkpoint recovery.
Highlights
For the cell to recover from the G2/M DNA damage checkpoint and resume cell cycle progression into mitosis, it needs to turn off checkpoint signaling and reactivate mitotic kinases that are required for mitotic progression
An important lesson learned from previous studies on Polo-like kinase 1 (Plk1) is that this mitotic kinase, known to be inhibited by checkpoint signaling, becomes reactivated during checkpoint recovery
Cells in which Plk1 is dispensable for mitotic entry during normal cell cycle fail to recover from the G2/M DNA damage checkpoint and to reenter mitosis without Plk1, suggesting a more stringent requirement of Plk1 for checkpoint recovery than for unperturbed cell cycle progression [7]
Summary
Polo-like kinase 1; CDK/Cdk, cyclin-dependent kinase; Gwl, Greatwall; KD, kinase-dead; MPF, maturation promoting factor; Plx, Xenopus Polo-like kinase 1. First identified in Drosophila and extensively studied in Xenopus egg extracts and human cells, Gwl has been shown to be an essential mitotic kinase activated by phosphorylation [15,16,17,18,19]. Gwl-dependent inhibition of PP2A/B55␦ is exerted via ␣-endosulfine (Ensa) or cAMP-regulated phosphoprotein, 19-kDa (Arpp-19), two related proteins that have been recently identified as key substrates of Gwl kinase in Xenopus egg extracts. Ensa and/or Arpp-19, once phosphorylated by Gwl, bind and inhibit PP2A/B55␦, yet spare PP2A complexed to other targeting subunits [25,26,27] These elegant studies have gone a long way in revealing Gwl functions, it is still largely unclear how Gwl activation is regulated or whether Gwl may function through other, as yet unknown substrates. We identify an indirect route of Plx regulation by Gwl during checkpoint recovery: Gwl does not directly phosphorylate Plx, but rather controls activation of Aurora A, which has been recently shown to serve as the upstream, T-loop kinase of Plx1 [12, 13]
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