Negative regulation of Chk2 expression by p53

在哺乳动物细胞中，激酶Chk2和肿瘤抑制因子P53参与了一种明确的调控相互作用。目前已证明，在Saos2细胞中，P53的诱导降低了Chk2的mRNA和蛋白的丰度。电离辐射还触发了人成纤维细胞中Chk2的磷酸化和随后的下调；对缺乏功能性P53的HeLa细胞的辐射诱导了Chk2的磷酸化，但不诱导其下调。A172细胞的报告基因分析表明，野生型p53抑制了人Chkk2基因启动子的活性，而显性阴性突变体则增强了人Chkk2基因启动子的活性。突变分析表明，位于启动子-152~-138之间的CCAAT盒是其受P53负调控的原因。凝胶迁移率分析表明，转录因子NF-Y与该CCAAT序列结合。核因子-Y的显性负突变消除了P53对Chk2启动子活性的影响。最后，在A172细胞中，在P53不敏感启动子的控制下，Chk2的表达延迟了电离辐射诱导的G2-M期停滞后细胞周期的重新进入。这些结果表明，P53通过调节核因子-Y功能来负向调节Chk2基因的转录，并且这种调节对于细胞在DNA损伤后重新进入细胞周期是重要的。

The kinase Chk2 and tumor suppressor p53 participate in an in-defined regulatory interaction in mammalian cells. The abundance of Chk2 mRNA and protein has now been shown to be decreased by the induction of p53 in Saos2 cells. Ionizing radiation also triggered the phosphorylation and subsequent down-regulation of Chk2 in human fibroblasts ; irradiation of HeLa cells, which lack functional p53, induced Chk2 phosphorylation but not its down-regulation. Reporter gene assays in A172 cells revealed that wild-type p53 repressed, whereas a dominant negative p53 mutant increased, the activity of the human Chkk2 gene promoter. Mutational analysis showed that a CCAAT box located between nucleotides -152〜-138 of the promoter was responsible for its negative regulation by p53. Electrophoretic mobility-shift assays demonstrated that the transcription factor NF-Y binds to this CCAAT sequence. A dominant negative mutant of NF-Y abolished the effect of p53 on Chk2 promoter activity. Finally, expression of Chk2 under the control of a p53-insensitive promoter in A172 cells delayed reentry into the cell cycle after G2-M arrest induced by ionizing irradiation. These results suggest that p53 negatively regulates Chk2 gene transcription through modulation of NF-Y function, and that this regulation is important for reentry of cells into the cell cycle after DNA damage.