Phosphorylation of serine 392 stabilizes the tetramer formation of tumor suppressor protein p53
Phosphorylation of serine 392 stabilizes the tetramer formation of tumor suppressor protein p53
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DOI：
10.1021/bi970759w
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发表时间：
1997-08-19
期刊：
影响因子：
2.9
通讯作者：
Xie, D
中科院分区：
文献类型：
--
作者：
Sakaguchi, K;Sakamoto, H;Xie, D
肿瘤抑制蛋白p53是一种四聚体磷蛋白，其响应于DNA损伤而激活来自若干细胞周期调控基因的转录。四聚体的形成对p53激活转录的能力至关重要;然而，翻译后修饰和蛋白质稳定化也有助于p53激活转录的能力。为了确定磷酸化是否影响四聚体形成，我们合成了对应于人p53残基303-393的磷酸肽，其包括负责四聚体形成的结构域。磷酸盐在Ser 315、Ser 378或Ser 392处以及在Ser 315和Ser 392处化学掺入。平衡超微结构分析表明，在Ser 392磷酸化增加可逆四聚体形成的关联常数近10倍。Ser 315或Ser 378的磷酸化对四聚体的形成几乎没有影响，但Ser 315的磷酸化在很大程度上逆转了Ser 392的磷酸化作用。量热法分析表明，磷酸化可能会影响亚基亲和力（和，反过来，DNA结合）由一个蛋白质驱动的过程，可能之间的C-末端残基和区域紧邻Ser 315。未磷酸化的p53 C-末端结构域的四聚体-单体转换的Kd被确定为类似于1-10 μ M。因此，在正常、未受损的细胞中，p53可能主要是单体的。通过Ser 392的磷酸化增强四聚体形成，再加上DNA损伤诱导的其核浓度的增加，可以提供一个开关，激活p53作为转录因子响应DNA损伤。
Tumor suppressor protein p53 is a tetrameric phosphoprotein that activates transcription from several cell cycle regulating genes in response to DNA damage. Tetramer formation is critical to p53's ability to activate transcription; however, posttranslational modifications and protein stabilization also contribute to p53's ability to activate transcription. To determine if phosphorylation affects tetramer formation, we synthesized phosphopeptides corresponding to residues 303-393 of human p53, which includes the domain responsible for tetramer formation, Phosphate was chemically incorporated at Ser315, Ser378, or Ser392 and also at both Ser315 and Ser392. Equilibrium ultracentrifugal analyses showed that phosphorylation at Ser392 increased the association constant for reversible tetramer formation nearly 10-fold. Phosphorylation of either Ser315 or Ser378 had little effect on tetramer formation, but phosphorylation of Ser315 largely reversed the effect of phosphorylation at Ser392. Analyses by calorimetry demonstrated that phosphorylation may influence subunit affinity (and, in turn, DNA binding) by an enthalpy-driven process, possibly between the C-terminal residues and the region immediately adjacent to Ser315. The K-d for the tetramer-monomer transition of the unphosphorylated p53 C-terminal domain was determined to be similar to 1-10 mu M. Thus, in normal, undamaged cells p53 may be largely monomeric. Enhancement of tetramer formation through phosphorylation of Ser392, coupled with a DNA-damage-induced increase in its nuclear concentration, could provide a switch that activates p53 as a transcription factor in response to DNA damage.