Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Center for Molecular Discovery, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Proc Natl Acad Sci U S A. 2010 Mar 9;107(10):4579-84. doi: 10.1073/pnas.0912094107. Epub 2010 Feb 19.
In unstressed cells, the tumor suppressor p53 is maintained at low levels by ubiquitin-mediated proteolysis mainly through Mdm2. In response to DNA damage, p53 is stabilized and becomes activated to turn on transcriptional programs that are essential for cell cycle arrest and apoptosis. Activation of p53 leads to accumulation of Mdm2 protein, a direct transcriptional target of p53. It is not understood how p53 is protected from degradation when Mdm2 is up-regulated. Here we report that p53 stabilization in the late phase after ionizing radiation correlates with active ubiquitination. We found that an E3 ubiquitin ligase RFWD3 (RNF201/FLJ10520) forms a complex with Mdm2 and p53 to synergistically ubiquitinate p53 and is required to stabilize p53 in the late response to DNA damage. This process is regulated by the DNA damage checkpoint, because RFWD3 is phosphorylated by ATM/ATR kinases and the phosphorylation mutant fails to stimulate p53 ubiquitination. In vitro experiments suggest that RFWD3 is a p53 E3 ubiquitin ligase and that RFWD3-Mdm2 complex restricts the polyubiquitination of p53 by Mdm2. Our study identifies RFWD3 as a positive regulator of p53 stability when the G(1) cell cycle checkpoint is activated and provides an explanation for how p53 is protected from degradation in the presence of high levels of Mdm2.
在非应激细胞中，肿瘤抑制因子 p53 通过泛素介导的蛋白水解作用被维持在低水平，主要通过 Mdm2 进行。响应于 DNA 损伤，p53 被稳定并被激活，从而开启对细胞周期停滞和细胞凋亡至关重要的转录程序。p53 的激活导致 Mdm2 蛋白的积累，Mdm2 是 p53 的直接转录靶标。目前尚不清楚当 Mdm2 上调时，p53 如何免受降解。在这里，我们报告说，电离辐射后晚期的 p53 稳定与活跃的泛素化相关。我们发现 E3 泛素连接酶 RFWD3（RNF201/FLJ10520）与 Mdm2 和 p53 形成复合物，协同泛素化 p53，并需要在 DNA 损伤的晚期反应中稳定 p53。这个过程受到 DNA 损伤检查点的调节，因为 RFWD3 被 ATM/ATR 激酶磷酸化，而磷酸化突变体不能刺激 p53 泛素化。体外实验表明，RFWD3 是 p53 的 E3 泛素连接酶，并且 RFWD3-Mdm2 复合物限制了 Mdm2 对 p53 的多泛素化。我们的研究确定 RFWD3 是 G1 细胞周期检查点激活时 p53 稳定性的正向调节剂，并为在高水平 Mdm2 存在下 p53 如何免受降解提供了解释。
