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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    • "Fig- ure 5Bshows that Swe1 is degraded with similar kinetics in undamaged CDC13 cells and in cdc13-1 mutants experiencing DNA damage. Taken together, these results indicate that phosphorylation of early mitotic substrates by Cdc5 is not downregulated during the DNA damage response.Hu et al., 2001;Liang and Wang, 2007;Schleker et al., 2010;Valerio-Santiago et al., 2013;Vidanes et al., 2010;Zhang et al., 2009). Among those, the Sae2 endonuclease stands out because of its importance in DNA damage signaling (Clerici et al., 2006;Donnianni et al., 2010). "
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    ABSTRACT: Cell-cycle checkpoints are essential feedback mechanisms that promote genome integrity. However, in the face of unrepairable DNA lesions, bypass mechanisms can suppress checkpoint activity and allow cells to resume proliferation. The molecular mechanisms underlying this biological response are currently not understood. Taking advantage of unique separation-of-function mutants, we show that the Polo-like kinase (PLK) Cdc5 uses a phosphopriming-based interaction mechanism to suppress G2/M checkpoint arrest by targeting Polo kinase activity to centrosomes. We also show that key subunits of the evolutionarily conserved RSC complex are critical downstream effectors of Cdc5 activity in checkpoint suppression. Importantly, the lethality and checkpoint defects associated with loss of Cdc5 Polo box activity can be fully rescued by artificially anchoring Cdc5 kinase domain to yeast centrosomes. Collectively, our results highlight a previously unappreciated role for centrosomes as key signaling centers for the suppression of cell-cycle arrest induced by persistent or unrepairable DNA damage.
    Full-text · Article · Jan 2016 · Cell Reports
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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.
    Full-text · Article · Oct 2014 · Cell Reports
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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.
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