Regulation of Function of p53 by Phosphorylation and Acetylation

通过磷酸化和乙酰化调节 p53 的功能

• 批准号: 11694336
• 负责人: TAYA Yoichi
• 金额: $ 1.73万
• 依托单位: NATIONAL CANCER CENTER RESEARCH INSTITUTE
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11694336/
• 关键词: p53; Phosphorylation; DNA replication; DNA damage; ATM; Chk2; Chk1; チェックポイントコントロール; Mdm2; L1-Fraumeni症候群

Upon DNA damage, the amino terminus of p53 is phosphorylated at a number of residues including S20, a site that is particularly important in regulating stability and function of the protein. Because no known kinase has been identified that can modify this site, HeLa nuclear extracts were fractionated and S20 phosphorylation was followed. We discovered that a S20 kinase activity copurifies with the human homolog of the Schizosaccharmyces pombe checkpoint kinase, Chk1 (hCHK1). We confirmed that recombinant hCHK1, but not a kinase-defective version of hCHK1, can phosphorylates p53 in vitro at S20. The human homolog of the second S.pombe checkpoint kinase, Cds1 (CHK2/hCds1), phosphorylates tetrameric p53 but not monomeric p53 in vitro at sites similar to those phosphorylated by hCHK1 kinase, suggesting that both checkpoint kinases can play roles in regulating p53 after DNA damage.P53 is required for the induction of a G1 and/or G2 irreversible arrest after gamma-irradiation, whereas blocked DNA replication causes a p53-independent S-phase arrest. We have examined the response to p53 when DNA synthesis is blocked by hydroxyurea or aphidicolin or when DNA is damaged by gamma-irradiation. Our results suggest that stalled replication forks activate kinases that modify and stabilize p53, yet act downstream of ATM to impair p53 transcriptional activity.

在DNA损伤时，p53的氨基末端在包括S20的许多残基处被磷酸化，S20是在调节蛋白质的稳定性和功能中特别重要的位点。因为没有已知的激酶已被确定，可以修改这个网站，HeLa细胞核提取物分级和S20磷酸化。我们发现S20激酶活性与粟酒裂殖酵母检查点激酶Chk 1（hCHK 1）的人类同源物共纯化。我们证实，重组hCHK 1，但不是一个激酶缺陷型的hCHK 1，可以磷酸化p53在体外S20。第二个粟酒裂殖酵母检查点激酶Cds 1的人类同源物（CHK 2/hCds 1）在体外磷酸化四聚体p53而不是单体p53，其位点与被hCHK 1激酶磷酸化的位点相似，这表明两种检查点激酶都可以在DNA损伤后调节p53中发挥作用。P53是诱导γ射线照射后G1和/或G2不可逆停滞所必需的，而阻断DNA复制导致不依赖于p53的S期阻滞。我们已经研究了当DNA合成被羟基脲或阿非迪霉素阻断或当DNA被γ射线损伤时对p53的反应。我们的研究结果表明，停滞的复制叉激活激酶，修改和稳定p53，但ATM下游的行为损害p53的转录活性。

项目成果

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