Regultion of p53 Deubiquitination

描述（由申请人提供）：p53是一种重要的肿瘤抑制因子，在超过50%的人类癌症中发生突变。p53的一个主要功能是激活其靶基因的表达，如响应细胞应激的p21和p53 A，从而调节细胞周期进程和细胞凋亡。p53活性主要受翻译后修饰的调节，如泛素化、类小泛素化、磷酸化、乙酰化和甲基化。其中，Mdm 2诱导的p53泛素化诱导p53核输出和降解，这是调节p53活性的主要细胞机制。然而，逆转p53泛素化的过程，即去泛素化，还没有很好地理解。我们已经发现，泛素特异性蛋白酶USP 10在体外和细胞中使p53去泛素化。在未应激的细胞和受到DNA损伤的细胞中，USP 10是稳定p53所必需的。USP 10主要定位于未应激细胞的细胞质中。响应于DNA损伤，USP 10在DNA损伤后稳定并易位到细胞核中，并且USP 10的ATM依赖性磷酸化对于USP 10的易位和稳定是重要的。此外，我们发现USP 10抑制具有野生型p53的细胞中的肿瘤细胞生长，同时促进具有突变型p53的细胞中的肿瘤细胞生长。最后，我们发现在高比例的肾细胞癌病例中USP 10表达缺失。基于这些初步结果，我们假设USP 10是p53的重要调节因子，USP 10通过p53抑制肿瘤发生。因此，重要的是进一步研究细胞和模型系统中USP 10-p53通路的调节。我们提出以下具体目标：1。研究Ras和G3 BP 1对USP 10-p53通路的调控。G3 BP 1（Ras-GTP酶激活蛋白SH 3-结构域结合蛋白）是Ras的下游效应子，先前已显示在体外抑制USP 10。我们假设Ras-G3 BP 1通路通过USP 10调节p53的稳定性，并将在此目的中检验这一假设。2.研究DNA损伤反应途径对USP 10的调控。USP 10主要定位于未应激细胞的细胞质中。然而，在DNA损伤后，USP 10易位到细胞核并在DNA损伤后变得稳定，这有助于p53激活。我们已经证明USP 10的ATM依赖性磷酸化是USP 10稳定和易位所必需的，然而这些事件背后的机制并不完全清楚。我们发现USP 10也以磷酸化依赖的方式被sumoylated，这表明USP 10 sumoylation可能对USP 10的稳定和易位很重要。我们将研究USP 10 SUMO化的功能意义和调控。3.研究USP 10在肿瘤发生中的作用。由于USP 10正调控p53，我们假设USP 10作为肿瘤抑制因子发挥作用。与这一假设一致，USP 10可以以p53依赖性方式抑制癌细胞生长，并且在高百分比的肾细胞癌样品中USP 10表达下调。我们将使用USP 10基因敲除小鼠作为模型系统，研究USP 10在体内肿瘤发生中的作用。总之，这些研究将揭示p53调控和肿瘤抑制的新机制。它们也将为未来的癌症分子干预治疗奠定基础。

DESCRIPTION (provided by applicant): p53 is an important tumor suppressor that is mutated in more than 50% of human cancers. A major function of p53 is to activate the expression of its target genes, such as p21 and PUMA in response to cellular stress, thereby regulating cell cycle progression and apoptosis. p53 activity is mainly regulated by posttranslational modifications, such as ubiquitination, sumoylation, phosphorylation, acetylation, and methylation. Among them, Mdm2-induced ubiquitination of p53 induces p53 nuclear export and degradation, which is a major cellular mechanism that regulates p53 activity. However, the process of reversing ubiquitination of p53, i.e. deubiquitination, is not well understood. We have found that the ubiquitin-specific protease USP10 deubiquitinates p53 in vitro and in cells. USP10 is required for the stabilization p53 in both unstressed cells and cells subjected to DNA damage. USP10 predominately localizes in the cytoplasm in unstressed cells. In response to DNA damage, USP10 is stabilized and translocates into the nucleus following DNA damage, and ATM-dependent phosphorylated of USP10 is important for USP10 translocation and stabilization. Furthermore, we found that USP10 suppresses tumor cell growth in cells with wild-type p53, while promoting tumor cell growth in cells with mutant p53. Finally, we found loss of USP10 expression in high percentage of renal cell carcinoma cases. Based on these preliminary results, we hypothesize that USP10 is an important regulator of p53, and USP10 suppresses tumorigenesis through p53. Therefore, it is important to further investigate the regulation of the USP10-p53 pathway in cells and in model systems. We propose the following Specific Aims: 1. Study the regulation of USP10-p53 pathway by Ras and G3BP1. G3BP1 (Ras- GTPase-activating protein SH3-domain-binding protein), a downstream effector of Ras, has previously been shown to inhibit USP10 in vitro. We hypothesize that the Ras-G3BP1 pathway regulates p53 stability through USP10, and will test this hypothesis in this aim. 2. Study the regulation of USP10 by the DNA damage response pathway. USP10 predominately localizes in the cytoplasm of unstressed cells. However, following DNA damage, USP10 translocates to the nucleus and becomes stabilized following DNA damage, which contributes to p53 activation. We have shown that ATM-dependent phosphorylation of USP10 is required for USP10 stabilization and translocation, however the mechanisms underlying these events are not entirely clear. We found that USP10 is also sumoylated in a phosphorylation-dependent manner, suggesting that USP10 sumoylation might be important for USP10 stabilization and translocation. We will study the functional significance and regulation of USP10 sumoylation in this aim. 3. Examine the role of USP10 in tumorigenesis. Since USP10 positively regulates p53, we hypothesize that USP10 functions as a tumor suppressor. Consistent with this hypothesis, USP10 can suppress cancer cell growth in p53-dependent manner and USP10 expression is downregulated in high percentage of renal cell carcinoma samples. We will examine the role of USP10 in tumorigenesis in vivo using USP10 knockout mice as a model system. In summary, these studies will reveal novel mechanisms of p53 regulation and tumor suppression. They will also lay the base for future molecular intervention for cancer therapy.