Wang Yaru, Zhou Xindi, Lin Zhiqiao, Huang Yichao, Zhang Yuchao, Liu Haobo, Zhou Yuqian, Liu Jianping, Pan Lifeng
School of Chemistry and Materials Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, 1 Sub-lane Xiangshan, Hangzhou, Zhejiang, China.
State Key Laboratory of Chemical Biology, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai, China.
Cell Discov. 2025 Sep 16;11(1):75. doi: 10.1038/s41421-025-00824-x.
HOIP, an RBR-type E3 ligase and the catalytic subunit of the linear ubiquitin chain assembly complex (LUBAC), plays crucial roles in various cellular processes, including the NF-κB signaling pathway. The E3 activity of HOIP can be inhibited by the kinase STK4-mediated phosphorylation, although the mechanism is poorly understood. In this study, using biochemical, mass spectrometry and structural approaches, we systemically characterize the association of STK4 with HOIP, and unveil that STK4 can directly bind to the RING2-LDD module of HOIP through its kinase domain. The determined crystal structure of STK4 in complex with HOIP RING2-LDD not only elucidates the detailed binding mechanism of STK4 with HOIP, but also uncovers, for the first time, a unique binding mode of STK4 with its substrate. Moreover, we reveal that STK4 can directly phosphorylate the T786 residue of HOIP that is located in the allosteric ubiquitin-binding site of HOIP. Importantly, the phosphorylation of HOIP T786 mediated by STK4 can block the binding of ubiquitin to the allosteric site of HOIP, thereby attenuating the E3 activity of HOIP. In all, our findings provide mechanistic insights into the interaction between STK4 and HOIP as well as the negative regulation of HOIP's E3 activity by STK4-mediated phosphorylation, which are valuable for further understanding the regulatory modes of RBR-type E3 ligases.
HOIP是一种RBR型E3连接酶，也是线性泛素链组装复合体（LUBAC）的催化亚基，在包括NF-κB信号通路在内的各种细胞过程中发挥关键作用。HOIP的E3活性可被激酶STK4介导的磷酸化作用抑制，尽管其机制尚不清楚。在本研究中，我们运用生化、质谱和结构学方法，系统地表征了STK4与HOIP的关联，并揭示STK4可通过其激酶结构域直接与HOIP的RING2-LDD模块结合。所测定的STK4与HOIP RING2-LDD复合物的晶体结构不仅阐明了STK4与HOIP的详细结合机制，还首次揭示了STK4与其底物的独特结合模式。此外，我们发现STK4可直接磷酸化HOIP位于变构泛素结合位点的T786残基。重要的是，STK4介导的HOIP T786磷酸化作用可阻断泛素与HOIP变构位点的结合，从而减弱HOIP的E3活性。总之，我们的研究结果为STK4与HOIP之间的相互作用以及STK4介导的磷酸化作用对HOIP E3活性的负调控提供了机制性见解，这对于进一步理解RBR型E3连接酶的调控模式具有重要价值。
