Wagner P, Fuchs A, Götz C, Nastainczyk W, Montenarh M
Medical Biochemistry, University of Saarland, Homburg/Saar, Germany.
Oncogene. 1998 Jan 8;16(1):105-11. doi: 10.1038/sj.onc.1201510.
In vivo p53 is multiply phosphorylated by different protein kinases suggesting a central role for phosphorylation in modulating p53 function. In addition, p53 was found to be associated with two protein kinases, p34cdc2 and protein kinase CK2. Here we report the precise mapping of the interaction sites of p53-p34cdc2 complexes. The p34cdc2 binding site on human p53 maps to one distinct C-terminal site LQIRGRERFE (aa 330-339) close to the corresponding phosphorylation site at serine 315. In order to test whether phosphorylation of p53 might influence the binding of p53 to p34cdc2 phosphorylation mutants of the C-terminus of p53, which mimick permanent phosphorylation, were tested on their ability to bind to p34cdc2 in vitro. Substitution of serine 315 (the p34cdc2 phosphorylation site) with aspartic acid had only little effect on complex formation whereas an exchange of serine 392 (the protein kinase CK2 phosphorylation site) to aspartic acid resulted in a significant reduced relative binding affinity of p53 to p34cdc2. The same result was obtained when the C-terminus of p53 was phosphorylated by purified protein kinase CK2 prior to examination of complex formation. In addition, the specificity of the complex formation has been checked by competition experiments with full length p53 proteins and the influence of cyclin B on complex formation was examined.
在体内，p53 被不同的蛋白激酶多重磷酸化，这表明磷酸化在调节 p53 功能中起核心作用。此外，发现 p53 与两种蛋白激酶 p34cdc2 和蛋白激酶 CK2 相关。在此我们报告 p53 - p34cdc2 复合物相互作用位点的精确图谱。人 p53 上的 p34cdc2 结合位点定位于一个独特的 C 末端位点 LQIRGRERFE（氨基酸 330 - 339），靠近丝氨酸 315 处的相应磷酸化位点。为了测试 p53 的磷酸化是否可能影响 p53 与 p34cdc2 的结合，对模拟永久磷酸化的 p53 C 末端磷酸化突变体在体外结合 p34cdc2 的能力进行了测试。将丝氨酸 315（p34cdc2 磷酸化位点）替换为天冬氨酸对复合物形成影响很小，而将丝氨酸 392（蛋白激酶 CK2 磷酸化位点）替换为天冬氨酸导致 p53 与 p34cdc2 的相对结合亲和力显著降低。在检查复合物形成之前，用纯化的蛋白激酶 CK2 对 p53 的 C 末端进行磷酸化时也得到了相同的结果。此外，通过与全长 p53 蛋白的竞争实验检查了复合物形成的特异性，并研究了细胞周期蛋白 B 对复合物形成的影响。
