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: 4.57). 01/2010; 9(2):350-63. DOI: 10.4161/cc.9.2.10448
Source: PubMed


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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    • "The positive control is from the experiment in Figure 1B (rad5Δ cells treated with 0.0033% MMS for 1 h after release from G1). The Rad53 doublet in G2/M was previously described and is not due to DNA damage-induced modification of this protein (Diani et al., 2009; Schleker et al., 2010; Tercero et al., 2003). "
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    ABSTRACT: The RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired lesions that block replication forks. It is subdivided into two branches: translesion DNA synthesis, which is frequently error prone, and the error-free DNA-damage-avoidance subpathway. Here, we show that Rad5(HLTF/SHPRH), which mediates the error-free branch, has a major role in the response to DNA damage caused by methyl methanesulfonate (MMS) during chromosome replication, whereas translesion synthesis polymerases make only a minor contribution. Both the ubiquitin-ligase and the ATPase/helicase activities of Rad5 are necessary for this cellular response. We show that Rad5 is required for the progression of replication forks through MMS-damaged DNA. Moreover, supporting its role during replication, this protein reaches maximum levels during S phase and forms subnuclear foci when replication occurs in the presence of DNA damage. Thus, Rad5 ensures the completion of chromosome replication under DNA-damaging conditions while minimizing the risk of mutagenesis, thereby contributing significantly to genome integrity maintenance.
    Cell Reports 10/2014; 9(2). DOI:10.1016/j.celrep.2014.09.005 · 8.36 Impact Factor
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    • "Cdc5/polo kinase is a crucial player in cell-cycle regulation from yeast to man, and the processes and substrates it regulates have been extensively investigated. Cdc5 regulates numerous cell cycle events, including promoting the destruction of the Cdk1-Clb2 inhibitor Swe1 [1], CLB2 cluster transcription (including itself) [2], spindle-pole body separation [3], spindle positioning [4], microtubule organization [5], recovery from hydroxyurea [6], APC/C activation [7], mitotic exit [8], cytokinesis [9,10], Cdc14 localization [11,12] and APC/CCdh1 inhibition [13]. However, the regulation of Cdc5 itself remains relatively uncharacterized. "
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    ABSTRACT: Cdc5 (polo kinase/Plk1) is a highly conserved key regulator of the S. cerevisiae cell cycle from S-phase until cytokinesis. However, much of the regulatory mechanisms that govern Cdc5 remain to be determined. Cdc5 is phosphorylated on up to 10 sites during mitosis. In this study, we investigated the function of phosphorylation site T23, the only full consensus Cdk1 (Cdc28) phosphorylation site present. Cdc5T23A introduces a degron that reduces its cellular amount to undetectable levels, which are nevertheless sufficient for normal cell proliferation. The degron acts in cis and is reversed by N-terminal GFP-tagging. Cdk1 kinase activity is required to maintain Cdc5 levels during G2. This, Cdk1 inhibited, Cdc5 degradation is APC/CCdh1 independent and requires new protein synthesis. Cdc5T23E is hyperactive, and reduces the levels of Cdc5 (in trans) and drastically reduces Clb2 levels. Phosphorylation of Cdc5 by Cdk1 is required to maintain Cdc5 levels during G2. However, phosphorylation of T23 (probably by Cdk1) caps Cdc5 and other CLB2 cluster protein accumulation, preventing potential protein toxicity, which may arise from their overexpression or from APC/CCdh1 inactivation.
    Cell Division 12/2011; 6(1):23. DOI:10.1186/1747-1028-6-23 · 3.53 Impact Factor
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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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    ABSTRACT: Defects in chromosome synapsis and/or meiotic recombination activate a surveillance mechanism that blocks meiotic cell cycle progression to prevent anomalous chromosome segregation and formation of aberrant gametes. In the budding yeast zip1 mutant, which lacks a synaptonemal complex component, the meiotic recombination checkpoint is triggered, resulting in extremely delayed meiotic progression. We report that overproduction of the polo-like kinase Cdc5 partially alleviates the meiotic prophase arrest of zip1, leading to the formation of inviable meiotic products. Unlike vegetative cells, we demonstrate that Cdc5 overproduction does not stimulate meiotic checkpoint adaptation because the Mek1 kinase remains activated in zip1 2μ-CDC5 cells. Inappropriate meiotic divisions in zip1 promoted by high levels of active Cdc5 do not result from altered function of the cyclin-dependent kinase (CDK) inhibitor Swe1. In contrast, CDC5 overexpression leads to premature induction of the Ndt80 transcription factor, which drives the expression of genes required for meiotic divisions, including CLB1. We also show that depletion of Cdc5 during meiotic prophase prevents the production of Ndt80 and that CDK activity contributes to the induction of Ndt80 in zip1 cells overexpressing CDC5. Our results reveal a role for Cdc5 in meiotic checkpoint control by regulating Ndt80 function.
    Molecular biology of the cell 07/2011; 22(18):3478-90. DOI:10.1091/mbc.E11-06-0482 · 4.47 Impact Factor
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