Regulation of Function of p53 by Phosphorylation and Acetylation

通过磷酸化和乙酰化调节 p53 的功能

基本信息

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

项目摘要

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.

DNA损伤后，p53的氨基末端在包括S20在内的许多残基上被磷酸化，S20在调节蛋白质的稳定性和功能方面尤为重要。由于尚未鉴定出可以修改该位点的已知激酶，因此将HELA核提取物分馏并遵循S20磷酸化。我们发现S20激酶活性与schizosaccharmyces Pombe检查点激酶（HCHK1）的人类同源物相关。我们证实了重组HCHK1，但不是HCHK1的激酶缺陷版本，可以在S20的体外磷酸化p53。第二s.pombe检查点激酶CDS1（CHK2/HCDS1）的人类同源物磷酸化，在与HCHK1激酶相似的位点上的四聚体p53磷酸化，而不是单体p53的体外p53，这两种检查点激酶都可以在dna saust for2 p53中扮演p53 suppul.p53，这两种检查点都可以扮演角色。 γ-辐照后不可逆的停滞，而阻塞的DNA复制会导致p53独立的S期停滞。当DNA合成被羟基脲或蚜虫蛋白阻断或DNA因γ-辐照受损时，我们检查了对p53的反应。我们的结果表明，停滞的复制叉激活了修饰和稳定p53的激酶，但在ATM下游作用以损害p53的转录活性。