Craig Ashley, Scott Mary, Burch Lindsay, Smith Graeme, Ball Kathryn, Hupp Ted
Department of Molecular and Cellular Pathology, Cancer Research UK Laboratories, University of Dundee, UK.
EMBO Rep. 2003 Aug;4(8):787-92. doi: 10.1038/sj.embor.embor901.
The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21(WAF1) and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21(WAF1) at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.
肿瘤抑制蛋白p53是一种四聚体蛋白，在DNA损伤时，其BOX-I反式激活结构域会被检查点激酶2（CHK2）磷酸化。CHK2不能磷酸化含有BOX-I基序的p53小肽片段，这表明p53四聚体中存在未明确的决定因素介导CHK2识别。源自p53 DNA结合结构域的两个肽与CHK2结合，并刺激全长p53在苏氨酸18和丝氨酸20处的磷酸化，从而确定了CHK2对接位点。这两个p53 DNA结合结构域肽可在反式作用中完全激活CHK2，并能使p53的BOX-I反式激活结构域片段在苏氨酸18和丝氨酸20处磷酸化。尽管CHK2具有基础的丝氨酸20激酶活性，主要被激活朝向苏氨酸18，但CHK1具有组成性的苏氨酸18激酶活性，主要在反式作用中被激活朝向丝氨酸20。CHK2对细胞分裂周期25C（CDC25C）的磷酸化不受p53 DNA结合结构域肽的影响。BOX-V基序中的CHK2对接位点是两个CHK2结合位点中最小的，在BOX-V肽中突变某些氨基酸会阻止CHK2激活。数据库搜索在p21（WAF1）中鉴定出一个p53 BOX-I同源基序，尽管CHK2对该蛋白无活性，但p53 DNA结合结构域肽可诱导p21（WAF1）在丝氨酸146处磷酸化。这提供了证据表明CHK2可通过一种新的对接相互作用对某些底物进行变构激活，并确定了一个潜在的调节开关，该开关可能在体内将CHK2引导至不同的信号通路。
