MDM2 is an E3 ubiquitin ligase that is able to ubiquitinate p53, targeting it for proteasomal degradation. Its homologue MDMX does not have innate E3 activity, but is able to dimerize with MDM2. Although mouse models have demonstrated both MDM2 and MDMX are individually essential for p53 regulation, the significance of MDM2-MDMX heterodimerization is only partially understood and sometimes controversial. MDM2C462A mice, where the C462A mutation abolishes MDM2 E3 ligase activity as well as its ability to dimerize with MDMX, die during embryogenesis. In contrast, the MDM2Y487A mice, where the Y487A mutation at MDM2 C-terminus significantly reduces its E3 ligase activity without disrupting MDM2-MDMX binding, survive normally even though p53 is expressed to high levels. This indicates that the MDM2-MDMX heterodimerization plays a critical role in the regulation of p53. However, it remains unclear whether MDMX is essential for the regulation of p53 protein levels in the context of an endogenous MDM2 C-terminal tail mutation. Here, we studied the significance of MDM2-MDMX binding in an MDM2 E3 ligase deficient context using the MDM2Y487A mouse embryonic fibroblast (MEF) cells. Surprisingly, down-regulation of MDMX in MDM2Y487A MEFs resulted in a significant increase of p53 protein levels. Conversely, ectopic overexpression of MDMX reduced p53 protein levels in MDM2Y487A MEFs. Mutations of the RING domain of MDMX prevented MDMX-MDM2 binding, and ablated MDMX-mediated suppression of p53 protein expression. Additionally, DNA damage treatment and nuclear sequestration of MDMX inhibited MDMX activity to suppress p53 protein expression. These results suggest that MDMX plays a key role in suppressing p53 protein expression in the absence of normal MDM2 E3 ligase activity. We found that the ability of MDMX to suppress p53 levels requires MDM2 binding and its cytoplasmic localization, and this ability is abrogated by DNA damage. Hence, MDMX is essential for the regulation of p53 protein levels in the context of an MDM2 C-terminal mutation that disrupts its E3 ligase activity but not MDMX binding. Our study is the first to examine the role of MDMX in the regulation of p53 in the context of endogenous MDM2 C-terminal mutant MEF cells.

中文翻译：

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在缺乏功能性 MDM2 C 末端尾部的情况下，MDMX 对于调节 p53 蛋白水平至关重要


MDM2 是一种 E3 泛素连接酶，能够泛素化 p53，靶向它进行蛋白酶体降解。其同源物 MDMX 不具有固有的 E3 活性，但能够与 MDM2 形成二聚体。尽管小鼠模型已证明 MDM2 和 MDMX 对于 p53 调节都是必需的，但 MDM2-MDMX 异二聚化的意义仅得到部分理解，有时甚至存在争议。 MDM2C462A 小鼠在胚胎发生过程中死亡，其中 C462A 突变消除了 MDM2 E3 连接酶活性及其与 MDMX 二聚化的能力。相比之下，MDM2Y487A 小鼠（其中 MDM2 C 末端的 Y487A 突变显着降低其 E3 连接酶活性，而不破坏 MDM2-MDMX 结合）即使 p53 高水平表达，也能正常存活。这表明MDM2-MDMX异二聚化在p53的调节中起着关键作用。然而，在内源性 MDM2 C 末端尾部突变的情况下，MDMX 是否对于 p53 蛋白水平的调节至关重要仍不清楚。在这里，我们使用 MDM2Y487A 小鼠胚胎成纤维细胞 (MEF) 研究了 MDM2 E3 连接酶缺陷环境中 MDM2-MDMX 结合的重要性。令人惊讶的是，MDM2Y487A MEF 中 MDMX 的下调导致 p53 蛋白水平显着增加。相反，MDMX 的异位过度表达降低了 MDM2Y487A MEF 中的 p53 蛋白水平。 MDMX RING 结构域的突变阻止了 MDMX-MDM2 结合，并消除了 MDMX 介导的 p53 蛋白表达抑制。此外，DNA 损伤处理和 MDMX 核隔离可抑制 MDMX 活性，从而抑制 p53 蛋白表达。这些结果表明，在缺乏正常 MDM2 E3 连接酶活性的情况下，MDMX 在抑制 p53 蛋白表达方面发挥着关键作用。 我们发现 MDMX 抑制 p53 水平的能力需要 MDM2 结合及其细胞质定位，而这种能力会因 DNA 损伤而消除。因此，MDMX 对于在 MDM2 C 末端突变的情况下调节 p53 蛋白水平至关重要，该突变会破坏其 E3 连接酶活性，但不会破坏 MDMX 结合。我们的研究首次在内源性 MDM2 C 端突变 MEF 细胞中检验 MDMX 在 p53 调节中的作用。