Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53.
Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53.
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DOI：
10.1101/gad.14.3.278
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发表时间：
2000-02
影响因子：
10.5
通讯作者：
N. Chehab;A. Malikzay;M. Appel;T. Halazonetis
中科院分区：
文献类型：
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作者：
N. Chehab;A. Malikzay;M. Appel;T. Halazonetis
Chk2/hcds1 是酿酒酵母 RAD53/SPK1 和粟酒裂殖酵母 cds1 DNA 损伤检查点基因的人类同源物，编码一种在 DNA 损伤后进行翻译后修饰的蛋白激酶。与其酵母同源物一样，Chk2/hCds1 蛋白在体外磷酸化 Cdc25C，这表明它可以响应 DNA 损伤而将细胞阻滞在 G(2) 状态。我们在人类细胞中表达 Chk2/hCds1 并分析了它们的细胞周期概况。 Chk2/hCds1 是野生型，但并非催化失活，在 DNA 损伤后导致 G(1) 停滞。显性失活 p53 突变体的共转染抑制了这种停滞，表明 Chk2/hCds1 在 p53 的上游发挥作用。在体外，Chk2/hCds1 在 Ser-20 上磷酸化 p53，并解离 p53 与 Mdm2（一种靶向 p53 降解的蛋白质）的预先形成的复合物。在体内，野生型 Chk2/hCds1 的异位表达导致 DNA 损伤后 p53 稳定性增强，而显性失活 Chk2/hCds1 突变体的表达消除了 p53 Ser-20 上的磷酸化和 p53 稳定性。因此，响应 DNA 损伤，Chk2/hCds1 稳定 p53 肿瘤抑制蛋白，导致细胞周期停滞在 G(1) 状态。
Chk2/hcds1, the human homolog of the Saccharomyces cerevisiae RAD53/SPK1 and Schizosaccharomyces pombe cds1 DNA damage checkpoint genes, encodes a protein kinase that is post-translationally modified after DNA damage. Like its yeast homologs, the Chk2/hCds1 protein phosphorylates Cdc25C in vitro, suggesting that it arrests cells in G(2) in response to DNA damage. We expressed Chk2/hCds1 in human cells and analyzed their cell cycle profile. Wild-type, but not catalytically inactive, Chk2/hCds1 led to G(1) arrest after DNA damage. The arrest was inhibited by cotransfection of a dominant-negative p53 mutant, indicating that Chk2/hCds1 acted upstream of p53. In vitro, Chk2/hCds1 phosphorylated p53 on Ser-20 and dissociated preformed complexes of p53 with Mdm2, a protein that targets p53 for degradation. In vivo, ectopic expression of wild-type Chk2/hCds1 led to increased p53 stabilization after DNA damage, whereas expression of a dominant-negative Chk2/hCds1 mutant abrogated both phosphorylation of p53 on Ser-20 and p53 stabilization. Thus, in response to DNA damage, Chk2/hCds1 stabilizes the p53 tumor suppressor protein leading to cell cycle arrest in G(1).