Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
J Biol Chem. 2011 Jul 8;286(27):23725-34. doi: 10.1074/jbc.M110.213868. Epub 2011 May 13.
Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.
遗传证据表明，Mdm2 和 MdmX 都是负调控 p53 的必需因子。然而，MdmX 在这种 Mdm2 依赖性蛋白降解中的确切作用还不是很清楚。大多数（如果不是全部）之前的 Mdm2 研究都在体外实验中使用 GST-Mdm2 融合蛋白。在这里，我们表明 GST-Mdm2 的 p53 多泛素化活性归因于 GST 标签，而非 GST 标记的 Mdm2 仅催化 p53 的单泛素化，即使在极高浓度下也是如此。我们进一步证明，MdmX 是 Mdm2 的一种有效的激活剂，促进了依赖剂量的 p53 多泛素化。这个激活过程需要 MdmX 和 Mdm2 蛋白的 RING 结构域。Mdm2/MdmX 的多泛素化活性依赖于 Mdm2。与单独过表达 Mdm2 或 MdmX 不同，MdmX 和 Mdm2 的共过表达一致地在细胞中触发 p53 降解。此外，只有在细胞质中才能观察到 p53 的细胞多泛素化，而细胞质中很容易检测到 Mdm2 和 MdmX。重要的是，RNAi 敲低 MdmX 会增加内源性 p53 的水平，并伴有 p53 多泛素化的减少。总之，我们的工作解决了由于使用 GST-Mdm2 而在该领域引起的主要混淆，并表明 MdmX 是将 Mdm2 从单泛素化 E3 连接酶转换为 p53 的多泛素化 E3 连接酶的细胞激活剂。总之，我们的研究结果为 Mdm2 和 MdmX 在 p53 稳定性的动态调节中的必要性提供了生化基础。
