The Notch signaling pathway, an important cell fate determination pathway, is modulated by the ubiquitin ligase Deltex. Here, we investigate the structural basis for Deltex-Notch interaction. We used nuclear magnetic resonance (NMR) spectroscopy to assign the backbone of the Drosophila Deltex WWE₂ domain and mapped the binding site of the Notch ankyrin (ANK) domain to the N-terminal WWE_A motif. Using cultured Drosophila S2R+ cells, we find that point substitutions within the ANK-binding surface of Deltex disrupt Deltex-mediated enhancement of Notch transcriptional activation and disrupt ANK binding in cells and in vitro. Likewise, ANK substitutions that disrupt Notch-Deltex heterodimer formation in vitro block disrupt Deltex-mediated stimulation of Notch transcription activation and diminish interaction with full-length Deltex in cells. Surprisingly, the Deltex-Notch intracellular domain (NICD) interaction is not disrupted by deletion of the Deltex WWE₂ domain, suggesting a secondary Notch-Deltex interaction. These results show the importance of the WWE_A:ANK interaction in enhancing Notch signaling.

Notch信号通路是重要的细胞命运决定通路，由泛素连接酶Deltex调节。在这里，我们研究了 Deltex-Notch 相互作用的结构基础。我们使用核磁共振 (NMR) 光谱来指定果蝇Deltex WWE ₂结构域的主链，并将 Notch 锚蛋白 (ANK) 结构域的结合位点映射到 N 端 WWE _A基序。使用培养的果蝇S2R+ 细胞，我们发现 Deltex 的 ANK 结合表面内的点替换破坏了 Deltex 介导的 Notch 转录激活增强，并破坏了细胞内和体外的ANK 结合。同样，在体外破坏Notch-Deltex异二聚体形成的ANK取代会破坏Deltex介导的Notch转录激活刺激，并减少与细胞中全长Deltex的相互作用。令人惊讶的是，Deltex-Notch 胞内结构域 (NICD) 相互作用并未因删除 Deltex WWE ₂结构域而中断，这表明存在二次 Notch-Deltex 相互作用。这些结果表明 WWE _A :ANK 相互作用在增强 Notch 信号传导方面的重要性。