Regultion of p53 Deubiquitination

描述(申请人提供)：P53是一种重要的肿瘤抑制基因，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p21和PUMA，以响应细胞应激，从而调节细胞周期进展和细胞凋亡。P53的活性主要受翻译后修饰的调节，如泛素化、苏莫化、磷酸化、乙酰化和甲基化。其中，MDM2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，还不是很清楚。我们已经在体外和细胞中发现泛素特异性蛋白水解酶USP10去泛素化P53。在非应激和DNA损伤的细胞中，USP10是稳定P53所必需的。在非应激细胞中，USP10主要定位于细胞质中。作为对DNA损伤的反应，USP10在DNA损伤后被稳定并移位到细胞核中，而ATM依赖的USP10磷酸化对USP10的移位和稳定是重要的。此外，我们发现USP10抑制野生型p53细胞的肿瘤细胞生长，而促进突变型P53细胞的肿瘤细胞生长。最后，我们发现USP10在高比例的肾癌中表达缺失。根据这些初步结果，我们假设USP10是P53的重要调节因子，并且USP10通过P53抑制肿瘤的发生。因此，进一步研究USP10-P53通路在细胞和模型系统中的调控是很重要的。1.研究RAS和G3BP1对USP10-P53通路的调节作用。G3BP1(RAS-GTP酶激活蛋白SH3结构域结合蛋白)是RAS的下游效应蛋白，已被证明在体外对USP10有抑制作用。我们假设Ras-G3BP1途径通过USP10调节P53的稳定性，并将在这一目标中检验这一假说。2.研究DNA损伤反应通路对USP10的调控作用。USP10主要定位于非应激细胞的细胞质中。然而，在DNA损伤后，USP10移位到细胞核，并在DNA损伤后变得稳定，这有助于P53的激活。我们已经证明，依赖ATM的USP10的磷酸化是USP10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，USP10也是以一种依赖于磷酸化的方式被求和的，这表明USP10求和对于USP10的稳定和易位可能是重要的。为了达到这个目的，我们将研究USP10苏莫化的功能意义和调控。3.检测USP10在肿瘤发生中的作用。由于USP10正向调节P53，我们假设USP10作为肿瘤抑制因子发挥作用。与这一假设一致，USP10可以P53依赖的方式抑制癌细胞的生长，并且USP10在高比例的肾癌样本中表达下调。我们将使用USP10基因敲除小鼠作为模型系统来研究USP10在体内肿瘤发生中的作用。综上所述，这些研究将揭示P53调控和肿瘤抑制的新机制。它们还将为未来癌症治疗的分子干预奠定基础。

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.