Pospísilová Sárka, Brázda Václav, Kucharíková Katerina, Luciani M Gloria, Hupp Ted R, Skládal Petr, Palecek Emil, Vojtesek Borivoj
Masaryk Memorial Cancer Institute, Zlutý kopec 7, CZ-656 53 Brno, Czech Republic.
Biochem J. 2004 Mar 15;378(Pt 3):939-47. doi: 10.1042/BJ20030662.
p53 is one of the most important regulators of cell proliferation and differentiation and of programmed cell death, triggering growth arrest and/or apoptosis in response to different cellular stress signals. The sequence-specific DNA-binding function of p53 protein can be activated by several different stimuli that modulate the C-terminal domain of this protein. The predominant mechanism of activation of p53 sequence-specific DNA binding is phosphorylation at specific sites. For example, phosphorylation of p53 by PKC (protein kinase C) occurs in undamaged cells, resulting in masking of the epitope recognized by monoclonal antibody PAb421, and presumably promotes steady-state levels of p53 activity in cycling cells. In contrast, phosphorylation by cdk2 (cyclin-dependent kinase 2)/cyclin A and by the protein kinase CK2 are both enhanced in DNA-damaged cells. We determined whether one mechanism to account for this mutually exclusive phosphorylation may be that each phosphorylation event prevents modification by the other kinase. We used non-radioactive electrophoretic mobility shift assays to show that C-terminal phosphorylation of p53 protein by cdk2/cyclin A on Ser315 or by PKC on Ser378 can efficiently stimulate p53 binding to DNA in vitro, as well as binding of the monoclonal antibody Bp53-10, which recognizes residues 371-380 in the C-terminus of p53. Phosphorylation of p53 by CK2 on Ser392 induces its DNA-binding activity to a much lower extent than phosphorylation by cdk2/cyclin A or PKC. In addition, phosphorylation by CK2 strongly inhibits PKC-induced activation of p53 DNA binding, while the activation of p53 by cdk2/cyclin A is not affected by CK2. The presence of CK2-mediated phosphorylation promotes PKC binding to its docking site within the p53 oligomerization domain, but decreases phosphorylation by PKC, suggesting that competition between CK2 and PKC does not rely on the inhibition of PKC-p53 complex formation. These results indicate the crucial role of p53 C-terminal phosphorylation in the regulation of its DNA-binding activity, but also suggest that antagonistic relationships exist between different stress signalling pathways.
p53是细胞增殖、分化及程序性细胞死亡最重要的调节因子之一，可响应不同的细胞应激信号触发生长停滞和/或凋亡。p53蛋白的序列特异性DNA结合功能可被几种不同的刺激激活，这些刺激调节该蛋白的C末端结构域。激活p53序列特异性DNA结合的主要机制是特定位点的磷酸化。例如，蛋白激酶C（PKC）介导的p53磷酸化发生在未受损细胞中，导致单克隆抗体PAb421识别的表位被掩盖，推测可促进循环细胞中p53活性的稳态水平。相反，细胞周期蛋白依赖性激酶2（cdk2）/细胞周期蛋白A及蛋白激酶CK2介导的磷酸化在DNA受损细胞中均增强。我们确定，解释这种相互排斥的磷酸化的一种机制可能是每个磷酸化事件都会阻止另一种激酶的修饰。我们使用非放射性电泳迁移率变动分析表明，cdk2/细胞周期蛋白A在Ser315处或PKC在Ser378处介导的p53蛋白C末端磷酸化可在体外有效刺激p53与DNA结合，以及识别p53 C末端371 - 380位残基的单克隆抗体Bp53 - 10的结合。CK2在Ser392处介导的p53磷酸化诱导其DNA结合活性的程度远低于cdk2/细胞周期蛋白A或PKC介导的磷酸化。此外，CK2介导的磷酸化强烈抑制PKC诱导的p53 DNA结合激活，而cdk2/细胞周期蛋白A对p53的激活不受CK2影响。CK2介导的磷酸化的存在促进PKC与其在p53寡聚化结构域内的对接位点结合，但降低了PKC介导的磷酸化，这表明CK2和PKC之间的竞争并不依赖于对PKC - p53复合物形成的抑制。这些结果表明p53 C末端磷酸化在调节其DNA结合活性中起关键作用，但也表明不同应激信号通路之间存在拮抗关系。
