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首页> 外文期刊>Cell cycle >Cell cycle-dependent phosphorylation of Rad53 kinase by Cdc5 and Cdc28 modulates checkpoint adaptation.
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Cell cycle-dependent phosphorylation of Rad53 kinase by Cdc5 and Cdc28 modulates checkpoint adaptation.

机译：Cdc5和Cdc28对Rad53激酶的细胞周期依赖性磷酸化可调节检查点适应性。

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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.
机译：在发芽酵母中，进化保守的检查点反应在整个细胞周期中对DNA破坏剂的敏感性不同。具体而言，激活S期细胞中的下游检查点激酶Rad53需要更多的损伤。我们在这里检查了是否通过细胞周期专用激酶的Rad53本身的磷酸化调节Rad53激活。我们发现，在不受干扰的生长过程中，Rad53在细胞周期的G（2），M和G（1）相中表现出小的磷酸化依赖性电泳迁移率移动，而在S相中丢失。我们显示Rad53在体外被Cdc5，有丝分裂的Polo样激酶和酵母细胞周期蛋白依赖性激酶Cdc28磷酸化。一致地，依赖细胞周期的Rad53迁移率变化需要Cdc5和Cdc28的活动。我们通过质谱图绘制了体外靶向的磷酸化位点，并通过质谱法证实Rad53内的丝氨酸774、789和791在M期停滞的细胞中被体内磷酸化。通过在内源性RAD53基因中创建不可磷酸化的突变，我们证实CDK和Polo激酶靶位点负责观察到的细胞周期依赖性蛋白迁移率变化。磷酸受体位点的丢失不会干扰Rad53的激活，但会在诱导单个不可修复的双链断裂后加速检查点的适应。因此，我们证明细胞周期依赖性磷酸化可以微调Rad53对DNA损伤的反应。

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