Genetic Analysis of p53 Stability and Activity

描述（由申请人提供）：肿瘤抑制基因p53的改变是人类癌症中最常见的突变。在各种应激反应中，p53蛋白水平及其活性被极大地诱导。然而，p53对各种应激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损伤和其他应激的反应中的生理作用仍有待确定。为了解决这个问题，我建议采用同源重组和LoxP-Cre介导的缺失引入错义突变（Ser/Thr到Ala突变）在几个潜在的重要的p53磷酸化位点，包括Ser 18和Thr 73/83，到内源性p53在小鼠。对p53（Ser 18 Ala）和p53（Thr 73/83 Ala）原代细胞的初步分析表明，这两种磷酸化事件在DNA损伤后调节p53稳定性和活性方面发挥重要但不同的作用。将确定这些p53基因敲入小鼠中p53对DNA损伤的反应受损的机制以及这些突变对p53依赖性肿瘤抑制的影响。此外，采用相同的方法，我们将确定潜在的功能冗余之间的磷酸化p53在Ser 18和ser 23调节p53对DNA损伤的反应。人p53丝氨酸46位的磷酸化被认为在调节p53凋亡功能中起重要作用。然而，人p53的Ser 46在小鼠p53中不保守。因此，将使用人p53基因敲入小鼠模型，其中小鼠p53基因的外显子4 -9被人p53基因的外显子4-9替换，以解决该磷酸化事件的生理作用。有两项意见表明了这一战略的可行性。首先，人源化p53在功能上等同于内源性小鼠p53。其次，DNA损伤诱导的信号通路导致人p53在Ser 46位的磷酸化在小鼠细胞中是保守的。鉴定调节p53稳定性和活性的磷酸化事件将指示所涉及的信号通路，从而揭示在各种应激或细胞衰老期间p53应答如何被激活的机制。

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.