Genetic Analysis of p53 Stability and Activity

描述(申请人提供)：肿瘤抑制基因p53的改变是人类癌症中最常见的突变。在对不同胁迫的反应中，P53蛋白水平和活性都被极大地诱导。然而，P53对各种压力的反应如何被激活的机制在很大程度上仍不清楚。最近的研究表明，P53的各种磷酸化事件可能调节P53的稳定性和活性。然而，这些P53磷酸化事件在调节P53对DNA损伤和其他应激反应中的生理作用仍有待确定。为了解决这个问题，我建议使用同源重组和loxP-Cre介导的缺失在小鼠的内源性P53中引入几个潜在的重要P53磷酸化位点的错义突变(Ser/Thr到Ala突变)，包括Ser18和Thr73/83。对P53(Ser18Ala)和P53(Thr73/83Ala)原代细胞的初步分析表明，这两个磷酸化事件在DNA损伤后调节P53的稳定性和活性方面发挥着重要但不同的作用。这些P53基因敲入小鼠对DNA损伤的反应受损的机制以及这些突变对P53依赖的肿瘤抑制的影响将被确定。此外，使用相同的方法，我们将确定在调节P53对DNA损伤的反应中，在Ser18和Ser23处的P53的磷酸化之间潜在的功能冗余。人类P53在Ser46位的磷酸化被认为在调节P53的凋亡功能中起重要作用。然而，人P53的SER46在小鼠P53中并不保守。因此，我们将用人P53基因的外显子4-9替换小鼠P53基因的外显子4-9的人P53基因敲入小鼠模型来研究这一磷酸化事件的生理作用。有两点意见表明了这一战略的可行性。首先，人源化的P53在功能上等同于内源性的小鼠P53。其次，DNA损伤诱导的导致人类P53在Ser46处磷酸化的信号通路在小鼠细胞中是保守的。识别调节P53稳定性和活性的磷酸化事件将揭示参与其中的信号通路，从而揭示P53在各种应激或细胞衰老过程中如何被激活的机制。

DESCRIPTION (provided by applicant): Alterations of the tumor suppressor p53 are the most commonly identified mutations in human cancers. In responses to various stresses, p53 protein level and its activity are greatly induced. However, the mechanism how p53 responses to various stresses are activated largely remains unclear. Recent studies have suggested that various phosphorylation events of p53 might regulate p53 stability and activity. However, the physiological roles of these phosphorylation events of p53 in regulating p53 responses to DNA damage and other stresses remain to be determined. To address this issue, I propose to employ homologous recombination and LoxP-Cre-mediated deletion to introduce missense mutations (Ser/Thr to Ala mutation) at several potentially important p53 phosphorylation sites, including Ser18 and Thr73/83, into the endogenous p53 in mice. Preliminary analysis of the p53(Ser18Ala) and p53(Thr73/83Ala) primary cells suggested that both phosphorylation events play important but distinct roles in regulating p53 stability and activity after DNA damage. The mechanism for the impaired p53 responses to DNA damage in these p53 knock-in mice and the effects of these mutations on the p53-dependent tumor suppression will be determined. In addition, employing the same approach, we will determine the potential functional redundancy between phosphorylation of p53 at Ser18 and ser23 in regulating p53 responses to DNA damage. Phosphorylation of human p53 at Ser46 has been suggested an important role in regulating p53 apoptotic function. However, Ser46 of human p53 is not conserved in mouse p53. Therefore, a human p53 knock-in mouse model, in which exons 4-9 of mouse p53 gene was replaced with exons 4-9 of human p53 gene, will be used to address the physiological roles of this phosphorylation event. Two observations indicated the feasibility of this strategy. First, the humanized p53 is functionally equivalent to the endogenous mouse p53. Secondly, the DNA damage-induced signaling pathways leading to the phosphorylation of human p53 at Ser46 is conserved in mouse cells. Identification of the phosphorylation events that regulate p53 stability and activity will indicate the signaling pathways involved and thus reveal the mechanism how p53 responses are activated during various stresses or cellular senescence.