Cell cycle-dependent phosphorylation of Rad53 kinase by Cdc5 and Cdc28 modulates checkpoint adaptation

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
Cell cycle (Georgetown, Tex.) (Impact Factor: 5.01). 01/2010; 9(2):350-63. DOI: 10.4161/cc.9.2.10448
Source: PubMed

ABSTRACT In budding yeast the evolutionarily conserved checkpoint response varies in its sensitivity to DNA damaging agents through the cell cycle. Specifically, higher amounts of damage are needed to activate the downstream checkpoint kinase Rad53 in S-phase cells. We examined here whether phosphorylation of Rad53 itself by cell cycle-dedicated kinases regulates Rad53 activation. We found that during unperturbed growth Rad53 exhibits a small phosphorylation-dependent electrophoretic mobility shift in G(2), M and G(1) phases of the cell cycle that is lost in S phase. We show that Rad53 is phosphorylated in vitro by Cdc5, a mitotic Polo-like kinase, and by the yeast cyclin-dependent kinase, Cdc28. Consistently, the cell cycle-dependent Rad53 mobility shift requires both Cdc5 and Cdc28 activities. We mapped the in vitro targeted phosphorylation sites by mass spectrometry and confirmed with mass spectroscopy that serines 774, 789 and 791 within Rad53 are phosphorylated in vivo in M-phase arrested cells. By creating nonphosphorylatable mutations in the endogenous RAD53 gene, we confirmed that the CDK and Polo kinase target sites are responsible for the observed cell cycle-dependent shift in protein mobility. The loss of phospho-acceptor sites does not interfere with Rad53 activation but accelerates checkpoint adaptation after induction of a single irreparable double-strand break. We thus demonstrate that cell cycle-dependent phosphorylation can fine-tune the response of Rad53 to DNA damage.

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Available from: Kenji Shimada, Oct 21, 2014
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    • "Cdc5 regulates this phenomenon, referred to as checkpoint adaptation, and the cdc5-ad allele is specifically defective in adaptation (Toczyski et al., 1997; Pellicioli et al., 2001). It was recently shown that high levels of Cdc5 counteract hyperphosphorylation of the effector DNA damage checkpoint kinase Rad53, thus resulting in a weakened checkpoint response and resumption of cell division despite the persistence of the checkpoint-inducing signal (Donnianni et al., 2010; Schleker et al., 2010; Vidanes et al., 2010). Because we observe a suppression of the checkpoint-dependent meiotic cell cycle block on CDC5 overexpression but the zip1 defects are still manifest, it was tempting to hypothesize that, as in mitotic cells, high levels of Cdc5 could be promoting meiotic checkpoint adaptation. "
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