Van Voorhis Vanessa A, Morgan David O
Department of Physiology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA.
Department of Physiology, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA.
Curr Biol. 2014 Jul 7;24(13):1556-62. doi: 10.1016/j.cub.2014.05.052. Epub 2014 Jun 12.
The anaphase-promoting complex/cyclosome (APC/C) is a protein-ubiquitin ligase (E3) that initiates the final events of mitosis by catalyzing the ubiquitination and proteasomal destruction of securin, cyclins, and other substrates [1, 2]. Like other members of the RING family of E3s [3, 4], the APC/C catalyzes direct ubiquitin transfer from an E2-ubiquitin conjugate (E2-Ub) to lysine residues on the protein substrate. The APC/C is activated at specific cell-cycle stages by association with an activator subunit, Cdc20 or Cdh1, which provides binding sites for specific substrate sequence motifs, or degrons. Activator might also stimulate catalytic activity [5, 6], but the underlying mechanisms are not known. Here, we dissected activator function using an artificial fusion substrate in which the N-terminal region of securin was linked to an APC/C core subunit. This fusion substrate bound tightly to the APC/C and was ubiquitinated at a low rate in the absence of activator. Ubiquitination of this substrate was stimulated by activator, due primarily to a dramatic stimulation of E2 sensitivity (Km) and catalytic rate (kcat), which together resulted in a 670-fold stimulation of kcat/Km. Thus, activator is not simply a substrate adaptor, but also enhances catalysis by promoting a more efficient interaction with the E2-Ub. Interestingly, full E2 stimulation required activator interaction with degron motifs on the substrate. We conclude that formation of a complete APC/C-activator-substrate complex leads to a major enhancement of E2 efficiency, providing an unusual substrate-assisted catalytic mechanism that limits efficient ubiquitin transfer to specific substrates.
后期促进复合物/细胞周期体（APC/C）是一种蛋白质泛素连接酶（E3），它通过催化securin、细胞周期蛋白和其他底物的泛素化及蛋白酶体降解来启动有丝分裂的最终事件[1,2]。与E3的RING家族的其他成员一样[3,4]，APC/C催化泛素从E2-泛素偶联物（E2-Ub）直接转移到蛋白质底物上的赖氨酸残基。APC/C在特定细胞周期阶段通过与激活亚基Cdc20或Cdh1结合而被激活，激活亚基为特定底物序列基序或降解子提供结合位点。激活剂也可能刺激催化活性[5,6]，但其潜在机制尚不清楚。在这里，我们使用一种人工融合底物来剖析激活剂的功能，其中securin的N端区域与APC/C核心亚基相连。这种融合底物与APC/C紧密结合，在没有激活剂的情况下以低速率被泛素化。激活剂刺激了该底物的泛素化，这主要是由于E2敏感性（Km）和催化速率（kcat）的显著刺激，两者共同导致kcat/Km增加了670倍。因此，激活剂不仅仅是底物适配器，还通过促进与E2-Ub更有效的相互作用来增强催化作用。有趣的是，完全的E2刺激需要激活剂与底物上的降解子基序相互作用。我们得出结论，完整的APC/C-激活剂-底物复合物的形成导致E2效率的显著提高，提供了一种不寻常的底物辅助催化机制，该机制将有效的泛素转移限制在特定底物上。
