Take Y, Kumano M, Teraoka H, Nishimura S, Okuyama A
Banyu Research Institute, Tsukuba, Japan.
Biochem Biophys Res Commun. 1996 Apr 16;221(2):207-12. doi: 10.1006/bbrc.1996.0575.
The tumor growth suppressor p21 has been shown to be induced by wild-type p53 (wt-p53) and to be a potent inhibitor of cyclin-dependent kinases and PCNA/DNA polymerase delta. Although wt-p53 is reported to be phosphorylated by several protein kinases, the function and significance of the phosphorylation of wt-p53 are not yet fully understood. Using OK-1035, a selective inhibitor of DNA-dependent protein kinase (DNA-PK), we demonstrated the importance of the phosphorylation of wt-p53 by DNA-PK in the DNA damage-mediated expression of the p21 gene. Treatment of HCT116, a human colon carcinoma cell line, with adriamycin induced the expression of wt-p53 and p21. By addition of OK-1035 to this culture, the induction of p21 protein was significantly decreased in a dose-dependent manner, whereas wt-p53 induction was not affected. Northern blot analysis revealed that suppression of p21 protein expression by OK-1035 resulted from reduction in the level of p21 mRNA. OK-1035 did not directly affect the binding ability of wt-p53 to its consensus DNA sequence. Our observations support the idea that wt-p53 induces the transcriptional activation of the p21 gene only after it is phosphorylated by DNA-PK.
肿瘤生长抑制因子p21已被证明可由野生型p53（wt-p53）诱导产生，并且是细胞周期蛋白依赖性激酶以及增殖细胞核抗原/DNA聚合酶δ的有效抑制剂。尽管据报道wt-p53可被多种蛋白激酶磷酸化，但其磷酸化的功能和意义尚未完全明确。我们使用DNA依赖性蛋白激酶（DNA-PK）的选择性抑制剂OK-1035，证明了DNA-PK介导的wt-p53磷酸化在DNA损伤介导的p21基因表达中的重要性。用阿霉素处理人结肠癌细胞系HCT116可诱导wt-p53和p21的表达。在此培养物中加入OK-1035后，p21蛋白的诱导表达以剂量依赖性方式显著降低，而wt-p53的诱导不受影响。Northern印迹分析显示，OK-1035对p21蛋白表达的抑制是由于p21 mRNA水平降低所致。OK-1035并不直接影响wt-p53与其共有DNA序列的结合能力。我们的观察结果支持这样一种观点，即wt-p53只有在被DNA-PK磷酸化后才会诱导p21基因的转录激活。
