Genetic Analysis of p53 Stability and Activity

描述（由申请人提供）：肿瘤抑制因子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蛋白水平及其活性受到极大的诱导。然而，p53对各种应激反应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，p53的这些磷酸化事件在调节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，我建议采用同源重组和loxp - cre介导的缺失，在几个潜在重要的p53磷酸化位点（包括Ser18和Thr73/83）引入错义突变（Ser/Thr到Ala突变）到小鼠内源性p53。对p53（Ser18Ala）和p53（Thr73/83Ala）原代细胞的初步分析表明，这两种磷酸化事件在DNA损伤后p53的稳定性和活性调节中发挥着重要但不同的作用。在这些p53敲入小鼠中，受损的p53对DNA损伤的反应机制以及这些突变对p53依赖性肿瘤抑制的影响将被确定。此外，采用相同的方法，我们将确定p53的Ser18和ser23磷酸化在调节p53对DNA损伤的反应中的潜在功能冗余。人p53第46位丝氨酸磷酸化已被认为在调节p53凋亡功能中起重要作用。然而，人类p53的Ser46在小鼠p53中并不保守。因此，人类p53敲入小鼠模型，将小鼠p53基因的外显子4-9替换为人类p53基因的外显子4-9，将用于解决这一磷酸化事件的生理作用。有两项意见表明这一战略的可行性。首先，人源化p53在功能上等同于内源性小鼠p53。其次，导致人类p53 Ser46位点磷酸化的DNA损伤诱导信号通路在小鼠细胞中是保守的。确定调节p53稳定性和活性的磷酸化事件将提示所涉及的信号通路，从而揭示在各种应激或细胞衰老过程中p53反应是如何被激活的机制。

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.