Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
Nat Chem Biol. 2012 Feb 26;8(4):383-92. doi: 10.1038/nchembio.801.
The anaphase-promoting complex/cyclosome (APC) is a ubiquitin ligase that is required for exit from mitosis. We previously showed that tosyl arginine methyl ester (TAME) inhibits APC-dependent proteolysis by competing with the C-terminal isoleucine-arginine tail of the APC activator cell division cycle 20 (Cdc20) for APC binding. Here we show that in the absence of APC substrates, TAME ejects Cdc20 from the APC by promoting Cdc20 autoubiquitination in its N-terminal region. Cyclin B1 antagonizes TAME's effect by promoting binding of free Cdc20 to the APC and by suppressing Cdc20 autoubiquitination. Nevertheless, TAME stabilizes cyclin B1 in Xenopus extracts by two mechanisms. First, it reduces the k(cat) of the APC-Cdc20-cyclin B1 complex without affecting the K(m), slowing the initial ubiquitination of unmodified cyclin B1. Second, as cyclin B1 becomes ubiquitinated, it loses its ability to promote Cdc20 binding to the APC in the presence of TAME. As a result, cyclin B1 ubiquitination terminates before reaching the threshold necessary for proteolysis.
后期促进复合物/细胞周期蛋白体（APC）是一种泛素连接酶，对于有丝分裂的退出是必需的。我们之前曾表明，甲苯磺酰精氨酸甲酯（TAME）通过与 APC 激活因子细胞分裂周期蛋白 20（Cdc20）的 C 末端异亮氨酸-精氨酸尾巴竞争 APC 结合，从而抑制 APC 依赖性蛋白水解。在这里，我们表明在没有 APC 底物的情况下，TAME 通过促进 Cdc20 的 N 端区域的自身泛素化，将 Cdc20 从 APC 中逐出。细胞周期蛋白 B1 通过促进游离 Cdc20 与 APC 的结合并抑制 Cdc20 自身泛素化来拮抗 TAME 的作用。尽管如此，TAME 通过两种机制稳定 Xenopus 提取物中的细胞周期蛋白 B1。首先，它降低了 APC-Cdc20-细胞周期蛋白 B1 复合物的 k(cat)，而不影响 K(m)，从而减缓了未修饰的细胞周期蛋白 B1 的初始泛素化。其次，随着细胞周期蛋白 B1 被泛素化，它失去了在 TAME 存在下促进 Cdc20 与 APC 结合的能力。结果，细胞周期蛋白 B1 的泛素化在达到蛋白水解所需的阈值之前就终止了。
