Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, 97080 Würzburg, Germany.
Cell Cycle, Biotechnology Center, Technische Universität Dresden, 01307 Dresden, Germany.
Sci Signal. 2020 Oct 20;13(654):eaba8208. doi: 10.1126/scisignal.aba8208.
At the heart of protein ubiquitination cascades, ubiquitin-conjugating enzymes (E2s) form reactive ubiquitin-thioester intermediates to enable efficient transfer of ubiquitin to cellular substrates. The precise regulation of E2s is thus crucial for cellular homeostasis, and their deregulation is frequently associated with tumorigenesis. In addition to driving substrate ubiquitination together with ubiquitin ligases (E3s), many E2s can also autoubiquitinate, thereby promoting their own proteasomal turnover. To investigate the mechanisms that balance these disparate activities, we dissected the regulatory dynamics of UBE2S, a human APC/C-associated E2 that ensures the faithful ubiquitination of cell cycle regulators during mitosis. We uncovered a dimeric state of UBE2S that confers autoinhibition by blocking a catalytically critical ubiquitin binding site. Dimerization is stimulated by the lysine-rich carboxyl-terminal extension of UBE2S that is also required for the recruitment of this E2 to the APC/C and is autoubiquitinated as substrate abundance becomes limiting. Consistent with this mechanism, we found that dimerization-deficient UBE2S turned over more rapidly in cells and did not promote mitotic slippage during prolonged drug-induced mitotic arrest. We propose that dimerization attenuates the autoubiquitination-induced turnover of UBE2S when the APC/C is not fully active. More broadly, our data illustrate how the use of mutually exclusive macromolecular interfaces enables modulation of both the activities and the abundance of E2s in cells to facilitate precise ubiquitin signaling.
在蛋白质泛素化级联反应的核心，泛素结合酶 (E2s) 形成反应性泛素硫酯中间体，以实现泛素向细胞底物的有效转移。因此，E2s 的精确调控对于细胞内稳态至关重要，它们的失调通常与肿瘤发生有关。除了与泛素连接酶 (E3s) 一起驱动底物泛素化外，许多 E2s 还可以自身泛素化，从而促进自身的蛋白酶体降解。为了研究平衡这些不同活性的机制，我们剖析了 UBE2S 的调控动态，UBE2S 是一种人类 APC/C 相关的 E2，可确保有丝分裂期间细胞周期调节剂的忠实泛素化。我们发现 UBE2S 的二聚体状态通过阻止催化关键的泛素结合位点来赋予自身抑制作用。二聚化受 UBE2S 的赖氨酸丰富的羧基末端延伸刺激，该延伸对于将该 E2 招募到 APC/C 也是必需的，并且当底物丰度变得有限时作为底物被自身泛素化。与该机制一致，我们发现二聚化缺陷的 UBE2S 在细胞中更快地周转，并且在长时间诱导的有丝分裂停滞期间不会促进有丝分裂滑溜。我们提出，当 APC/C 不完全活跃时，二聚化会减弱自身泛素化诱导的 UBE2S 周转。更广泛地说，我们的数据说明了如何使用相互排斥的大分子界面来调节细胞中 E2s 的活性和丰度，以促进精确的泛素信号传递。
