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p53参与了一种不确定的调节相互作用。在Saos2细胞中，由于p53的诱导，Chk2 mRNA和蛋白的丰度已经被证明是降低的。电离辐射也触发了人成纤维细胞中Chk2的磷酸化和随后的下调；辐照缺乏功能性p53的HeLa细胞，可诱导Chk2磷酸化，但不能诱导其下调。在A172细胞中的报告基因检测显示，野生型p53抑制了人类Chkk2基因启动子的活性，而显性阴性p53突变体则增加了人类Chkk2基因启动子的活性。突变分析表明，位于启动子-152 ~ -138核苷酸之间的CCAAT盒子负责p53对其进行负调控。电泳迁移转移实验表明转录因子NF-Y与该CCAAT序列结合。NF-Y显性负突变体消除了p53对Chk2启动子活性的影响。最后，在p53不敏感启动子的控制下，A172细胞中Chk2的表达延迟了电离照射诱导的G2-M阻滞后重新进入细胞周期。这些结果表明，p53通过调节NF-Y功能负向调节Chk2基因的转录，并且这种调节对于DNA损伤后细胞重新进入细胞周期非常重要。

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.