Wesierska-Gadek J, Schmid G, Cerni C
Institute of Tumorbiology-Cancer Research, University of Vienna, Austria.
Biochem Biophys Res Commun. 1996 Jul 5;224(1):96-102. doi: 10.1006/bbrc.1996.0990.
We have recently reported that mutant but not wild-type (wt) p53 protein was ADP-ribosylated in primary rat cells overexpressing the temperature-sensitive murine p53val135 gene. To examine whether the lack of susceptibility to modification is a specific feature of p53val135 adopting wt conformation or rather a general property of this tumor suppressor protein, we have studied ADP-ribosylation of wt p53 of different origin in vitro using semi-purified poly(ADP-ribose) transferase (pADPRT). In vitro pADPRT modified human and mouse wt p53 and p53val135. Under limiting substrate concentration, the molar mass of ADP-ribosylated p53 was only slightly altered. Chase experiments with high NAD concentration resulted in the formation of poly(ADP-ribosyl)ated p53 protein shifted to 64 kD. However, preincubation of wt p53 proteins with a p53 consensus sequence resulting in complex formation abolished the modification of wt p53. This indicates that in the cellular environment the specific DNA binding of wt p53 prevents its covalent modification by poly(ADP-ribose).
我们最近报道，在过表达温度敏感型鼠源p53val135基因的原代大鼠细胞中，突变型而非野生型（wt）p53蛋白发生了ADP核糖基化。为了研究对修饰不敏感是p53val135采用wt构象的特定特征，还是这种肿瘤抑制蛋白的普遍特性，我们使用半纯化的聚（ADP - 核糖）转移酶（pADPRT）在体外研究了不同来源的wt p53的ADP核糖基化。体外pADPRT修饰了人和小鼠的wt p53以及p53val135。在底物浓度有限的情况下，ADP核糖基化p53的摩尔质量仅略有改变。用高浓度NAD进行追踪实验导致形成了迁移至64 kD的聚（ADP - 核糖基）化p53蛋白。然而，将wt p53蛋白与p53共有序列预孵育导致形成复合物，从而消除了wt p53的修饰。这表明在细胞环境中，wt p53的特异性DNA结合可防止其被聚（ADP - 核糖）共价修饰。
