Department of Biological Sciences, Columbia University, New York, NY, USA.
Cell Cycle. 2010 Oct 1;9(19):3956-64. doi: 10.4161/cc.9.19.13162. Epub 2010 Oct 26.
The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction following DNA damage. To gain further insight into the processes regulating E2F1 protein stability, we focused our investigation on the mechanisms responsible for regulating E2F1 turnover. Here we show that E2F1 is a substrate of the anaphase promoting complex or cyclosome (APC/C), a ubiquitin ligase that plays an important role in cell cycle progression. Ectopic expression of the APC/C activators Cdh1 and Cdc20 reduced the levels of co-expressed E2F-1 protein. Co-expression of DP1 with E2F1 blocked APC/C-induced E2F1 degradation, suggesting that the E2F1/DP1 heterodimer is protected from APC/C regulation. Following Cdc20 knockdown, E2F1 levels increased and remained stable in extracts over a time course, indicating that APC/C(Cdc20) is a primary regulator of E2F1 stability in vivo. Moreover, cell synchronization experiments showed that siRNA directed against Cdc20 induced an accumulation of E2F1 protein in prometaphase cells. These data suggest that APC/C(Cdc20) specifically targets E2F1 for degradation in early mitosis and reveal a novel mechanism for limiting free E2F1 levels in cells, failure of which may compromise cell survival and/or homeostasis.
控制 E2F-1 活性的机制很复杂。我们之前曾表明，在 DNA 损伤后，Chk1 和 Chk2 对于 E2F1 的稳定和 p73 靶基因的诱导是必需的。为了更深入地了解调节 E2F1 蛋白稳定性的过程，我们将研究重点放在调节 E2F1 周转率的机制上。在这里，我们表明 E2F1 是后期促进复合物或细胞周期蛋白（APC/C）的底物，APC/C 是一种在细胞周期进程中起重要作用的泛素连接酶。APC/C 激活剂 Cdh1 和 Cdc20 的异位表达降低了共表达的 E2F-1 蛋白水平。E2F1 与 DP1 共表达可阻止 APC/C 诱导的 E2F1 降解，表明 E2F1/DP1 异二聚体免受 APC/C 调节。在 Cdc20 敲低后，E2F1 水平在提取物中随时间推移而增加并保持稳定，表明 APC/C(Cdc20) 是体内 E2F1 稳定性的主要调节因子。此外，细胞同步化实验表明，针对 Cdc20 的 siRNA 可在有丝前期细胞中诱导 E2F1 蛋白积累。这些数据表明，APC/C(Cdc20) 特异性地将 E2F1 作为早期有丝分裂中降解的靶标，并揭示了一种限制细胞中游离 E2F1 水平的新机制，该机制的失败可能会损害细胞存活和/或体内平衡。
