The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites.
The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites.
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DOI：
10.1101/gad.14.3.289
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发表时间：
2000-02
影响因子：
10.5
通讯作者：
S. Shieh;Jinwoo Ahn;K. Tamai;Y. Taya;C. Prives
中科院分区：
文献类型：
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作者：
S. Shieh;Jinwoo Ahn;K. Tamai;Y. Taya;C. Prives
在DNA损伤时，p53的氨基末端在包括S20的许多丝氨酸残基处被磷酸化，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。p53中另外的可诱导的氨基和羧基末端位点也被hCHK 1磷酸化，表明这是一种异常通用的蛋白激酶。有趣的是，hCHK 1在体外强烈偏好四聚体而不是单体p53，这与我们的观察结果一致，即体内氨基末端位点的磷酸化需要p53是寡聚体。转染细胞中hCHK 1的水平和活性的调节与p53的水平直接相关;激酶缺陷型hCHK 1或反义hCHK 1的表达导致共转染的p53水平降低，而野生型hCHK 1或hCHK 1的激酶结构域的过表达导致表达的p53蛋白水平增加。第二个S.粟酒裂殖酵母检查点激酶Cds 1（CHK 2/hCds 1）在体外磷酸化四聚体p53，但在与hCHK 1激酶磷酸化的位点相似的位点上不磷酸化单体p53，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。
Upon DNA damage, the amino terminus of p53 is phosphorylated at a number of serine 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 Schizosaccharomyces pombe checkpoint kinase, Chk1 (hCHK1). We confirmed that recombinant hCHK1, but not a kinase-defective version of hCHK1, can phosphorylate p53 in vitro at S20. Additional inducible amino- and carboxy-terminal sites in p53 are also phosphorylated by hCHK1, indicating that this is an unusually versatile protein kinase. It is interesting that hCHK1 strongly prefers tetrameric to monomeric p53 in vitro, consistent with our observation that phosphorylation of amino-terminal sites in vivo requires that p53 be oligomeric. Regulation of the levels and activity of hCHK1 in transfected cells is directly correlated with the levels of p53; expression of either a kinase-defective hCHK1 or antisense hCHK1 leads to reduced levels of cotransfected p53, whereas overexpression of wild-type hCHK1 or the kinase domain of hCHK1 results in increased levels of expressed p53 protein. 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.