Melchionna R, Chen X B, Blasina A, McGowan C H
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Nat Cell Biol. 2000 Oct;2(10):762-5. doi: 10.1038/35036406.
In response to DNA damage, eukaryotic cells use a system of checkpoint controls to delay cell-cycle progression. Checkpoint delays provide time for repair of damaged DNA before its replication in S phase and before segregation of chromatids in M phase. The Cds1 (Chk2) tumour-suppressor protein has been implicated in certain checkpoint responses in mammalian cells. It directly phosphorylates and inactivates the mitosis-inducing phosphatase Cdc25 in vitro and is required to maintain the G2 arrest that is observed in response to gamma-irradiation. Cds1 also directly phosphorylates p53 in vitro at a site that is implicated in its stabilization, and is required for stabilization of p53 and induction of p53-dependent transcripts in vivo upon gamma-ionizing radiation. Thus, Cds1 functions in both the G1 and G2 checkpoint responses. Like Cds1, the checkpoint protein kinase ATM (ataxia-telangiectasia-mutated) is required for correct operation of both the G1 and G2 damage checkpoints. ATM is necessary for phosphorylation and activation of Cds1 in vivo and can phosphorylate Cds1 in vitro, although evidence that the sites that are phosphorylated by ATM are required for activation is lacking. Here we show that threonine 68 of Cds1 is the preferred site of phosphorylation by ATM in vitro, and is the principal irradiation-induced site of phosphorylation in vivo. The importance of this phosphorylation site is demonstrated by the failure of a mutant, non-phosphorylatable form of Cds1 to be fully activated, and by its reduced ability to induce G1 arrest in response to ionising radiation.
真核细胞会利用一种检查点控制系统来延迟细胞周期进程，以应对DNA损伤。检查点延迟为受损DNA在S期复制前以及在M期染色单体分离前的修复提供了时间。Cds1（Chk2）肿瘤抑制蛋白参与了哺乳动物细胞的某些检查点反应。它在体外直接磷酸化并使诱导有丝分裂的磷酸酶Cdc25失活，并且是维持γ射线照射后观察到的G2期阻滞所必需的。Cds1在体外还能在一个与p53稳定有关的位点直接磷酸化p53，并且在体内γ电离辐射后p53的稳定以及p53依赖性转录本的诱导方面是必需的。因此，Cds1在G1和G2检查点反应中均发挥作用。与Cds1一样，检查点蛋白激酶ATM（共济失调毛细血管扩张症突变型）对于G1和G2损伤检查点的正确运行都是必需的。ATM在体内对于Cds1的磷酸化和激活是必要的，并且在体外能够磷酸化Cds1，尽管缺乏证据表明ATM磷酸化的位点对于激活是必需的。我们在此表明，Cds1的苏氨酸68是ATM在体外磷酸化的首选位点，并且是体内辐射诱导的主要磷酸化位点。一种不可磷酸化的Cds1突变体无法被完全激活，并且其响应电离辐射诱导G1期阻滞的能力降低，这证明了该磷酸化位点的重要性。
