Oncogenic Function of a P53-Induced Phosphatase

描述（由申请人提供）：肿瘤抑制因子p53在大多数人类肿瘤中在结构上或功能上失活。在那些保留结构完整的p53的肿瘤中，p53信号转导通常通过改变p53调节因子（如Mdm 2或ARF）的表达而激活。我们一直在研究编码丝氨酸/苏氨酸磷酸酶Wip 1（也称为PPM 1D）的p53转录靶点。与Mdm 2和ARF一样，Wip 1似乎也调节p53的功能。我们已经证明Wip 1通过多种机制抑制p53功能，包括（1）去磷酸化和抑制p53激酶，（2）p53本身的去磷酸化，和（3）去磷酸化和Mdm 2的稳定，这导致p53降解。除了对p53的抑制作用外，我们还发现Wip 1可使许多在细胞DNA损伤反应中被ATM和ATR激酶磷酸化的蛋白质去磷酸化。我们假设Wip 1作为DNA损伤反应的稳态调节剂，一旦损伤修复，通过促进受损细胞恢复到应激前的状态。Wip 1对p53的负作用使Wip 1成为一个明显的癌基因候选者。事实上，乳腺癌和其他几种人类癌症类型显示Wip 1基因的扩增和过表达。此外，Wip 1在啮齿动物成纤维细胞转化试验中作为致癌基因，我们已经证明缺乏Wip 1的小鼠对肿瘤具有抗性。为了更好地了解Wip 1在肿瘤发生和DNA损伤反应调节中的功能，我们提出了三个具体目标。目的1将集中在Wip 1的结构/功能的关系，通过诱变确定Wip 1的催化位点，并通过检查Wip 1/靶蛋白相互作用域。目的2需要识别和功能特性的新的Wip 1目标的ATM/ATR介导的DNA损伤反应中的重要。最后，Aim 3将使用基因工程小鼠模型来检查ATM/Wip 1和p53/Wip 1相互作用以及Wip 1介导的体内基因组完整性。公共卫生相关性：我们建议研究p53调控的癌基因Wip 1的功能，Wip 1在细胞周期控制和DNA损伤反应中起着重要作用。Wip 1是一种致癌的磷酸酶，部分原因是它抑制了p53的功能。具体来说，我们计划研究Wip 1的结构，确定Wip 1的新靶点，它如何与这些靶点相互作用，以及这种相互作用的功能后果。最后，我们想了解Wip 1如何影响完整哺乳动物（小鼠）的生理过程。

DESCRIPTION (provided by applicant): The tumor suppressor p53 is structurally or functionally inactivated in most human tumors. In those tumors that retain structurally intact p53, p53 signaling is often activated through altered expression of p53 regulators, such as Mdm2 or ARF. We have been investigating a p53 transcription target that encodes the serine/threonine phosphatase Wip1 (also known as PPM1D). Like Mdm2 and ARF, Wip1 appears to regulate p53 function. We have shown that Wip1 inhibits p53 function through multiple mechanisms, including (1) dephosphorylation and inhibition of p53 kinases, (2) dephosphorylation of p53 itself, and (3) dephosphorylation and stabilization of Mdm2, which leads to p53 degradation. In addition to its inhibitory effects on p53, we have also shown that Wip1 dephosphorylates a number of proteins that are phosphorylated by the ATM and ATR kinases in the cellular DNA damage response. We hypothesize that Wip1 acts as a homeostatic regulator of the DNA damage response by facilitating the return of the damaged cell back to a pre-stress state once damage is repaired. The negative effects of Wip1 on p53 make Wip1 an obvious oncogene candidate. In fact, breast cancers and several other human cancer types display amplification and overexpression of the Wip1 gene. Moreover, Wip1 acts as an oncogene in rodent fibroblast transformation assays and we have shown that mice lacking Wip1 are resistant to tumors. To better understand Wip1 function in oncogenesis and regulation of the DNA damage response, we propose three specific aims. Aim 1 will focus on Wip1 structure/function relationships through mutagenic ascertainment of the Wip1 catalytic site and by examination of Wip1/target protein interaction domains. Aim 2 entails the identification and functional characterization of novel Wip1 targets important in the ATM/ATR-mediated DNA damage response. Finally, Aim 3 will use genetically engineered mouse models to examine ATM/Wip1 and p53/Wip1 interactions as well as Wip1-mediated genomic integrity in an in vivo context. PUBLIC HEALTH RELEVANCE: We propose to investigate the functions of a p53 regulated oncogene, Wip1, that plays an important role in cell cycle control and the DNA damage response. Wip1 is a phosphatase that is oncogenic in part because it inhibits p53 function. Specifically, we plan to study the structure of Wip1, identify new targets of Wip1, how it interacts with those targets, and the functional consequences of such interactions. Finally, we would like to understand how Wip1 effects physiological processes in an intact mammal, the mouse.