Regulation of Function of p53 by Phosphorylation and Acetylation

在DNA损伤时，P53的氨基末端在包括S20在内的许多残基上被磷酸化，S20是调节蛋白质稳定性和功能的一个特别重要的位置。因为还没有发现可以修饰这个位点的已知的激酶，所以对HeLa核提取物进行了分级，然后进行了S20的磷酸化。我们发现S20激酶活性与人的裂殖酵母Pombe检查点激酶Chk1(HCHK1)的同源物相同。我们在S20证实了重组的hCHK1，但不是激酶缺陷版本的hCHK1，可以在体外磷酸化P53。第二个S.pombe检查点激酶的人类同源物CDs1(Chk2/hCDs1)在体外磷酸化四聚体P53，而不是单体P53，位置与hCHK1激酶磷酸化的位置相似，这表明这两个检查点激酶在DNA损伤后都可以在调节P53中发挥作用。P53是伽玛射线照射后诱导G1和/或G2不可逆停滞所必需的，而阻断DNA复制会导致P53非依赖于P53的S期停滞。我们研究了当DNA合成被羟基脲或aphidicolin阻止时，或者当DNA被伽马射线损伤时，对p53的反应。我们的结果表明，停滞不前的复制分叉激活了修改和稳定P53的激酶，但作用于ATM下游以削弱P53的转录活性。

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.