Human tumor-derived p53 proteins exhibit binding site selectivity and temperature sensitivity for transactivation in a yeast-based assay
Human tumor-derived p53 proteins exhibit binding site selectivity and temperature sensitivity for transactivation in a yeast-based assay
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人肿瘤来源的 p53 蛋白在基于酵母的检测中表现出反式激活的结合位点选择性和温度敏感性
作者：
C. Como;C. Prives
p53是具有许多已知靶基因的序列特异性转录激活因子，所述靶基因含有p53应答元件。在超过一半的人类肿瘤中，已经在其序列特异性DNA结合结构域中鉴定出p53的突变，尽管肿瘤衍生的p53突变体的子集保留了在某些条件下结合DNA和激活转录的能力。为了扩大我们对这种反式激活能力的理解，我们研究了p53突变体在基于酵母的测定中结合并激活报告基因的功效。对19个人肿瘤来源的p53突变体的分析，跨越p53的DNA结合结构域并包括热点类，揭示了在24 ℃、30 ℃和37 ℃的广泛的转录反式激活能力，尽管事实上每个突变体最初被鉴定为在酵母中对单个p53响应性RGC位点的报告基因无反式激活活性。一类突变体（P177 L、R267 W、C277 Y和R283 H）即使在生理温度（37 ℃）下也保持野生型或接近野生型的结合位点选择性活性。另一类突变体（V143 A、M160 I/A161 T、H193 R、Y220 C和I254 F），均定位于维持p53的β-支架，也保留了选择活性，但优先在亚生理温度（24和30 ℃）下。然而，引人注目的是，与其他肿瘤衍生的突变体相反，所有先前鉴定的热点突变体在所有测试的位点上都是完全无活性的。此外，一个双突变体，L22 E/W23 S，位于激活区域内，以前被证明是在成纤维细胞中转录失活，保留野生型或接近野生型结合位点选择性活性的酵母。最后，我们发现，在体内的转录活性并不一定与DNA结合在体外。
p53 is a sequence-specific transcriptional activator with a number of known target genes which contain p53-responsive elements. Mutations in p53 have been identified within its sequence-specific DNA binding domain in more than half of all human tumors, although a subset of tumor-derived p53 mutants have retained the ability to bind DNA and activate transcription under certain conditions. In order to broaden our understanding of this transactivating ability, we examined the efficacy by which p53 mutants bind to and activate reporters in an Saccharomyces cerevisiae-based assay. Analysis of 19 human tumor-derived p53 mutants, spanning the DNA binding domain of p53 and including thehot-spot'class, revealed a broad array of transcriptional transactivation abilities at 24 C, 30 C and 37 C, despite the fact that each mutant had originally been identified as being inactive for transactivation in yeast against a single p53-responsive RGC site-containing reporter. One class of mutants (P177L, R267W, C277Y and R283H) retained wild-type or near wild-type activity that is binding site-selective, even at physiological temperature (37 C). Another class of mutants (V143A, M160I/A161T, H193R, Y220C and I254F), all positioned for maintaining the β-scaffold of p53, also retained selective activity, but preferentially at sub-physiological temperatures (24 and 30 C). Strikingly, however, in contrast to the other tumor derived mutants, all of the previously identifiedhot-spot'mutants were completely inactive with all sites tested. Moreover, a double mutant, L22E/W23S, located within the activation region and previously shown to be transcriptionally inactive in fibroblasts, retained wild-type or near wild-type binding site-selective activity in yeast. Finally, we found that transcriptional activity in vivo does not necessarily correlate with DNA binding in vitro.