Wnt signals bind to Frizzled receptors to trigger canonical and noncanonical signaling responses that control cell fates during animal development and tissue homeostasis. All Wnt signals are relayed by the hub protein Dishevelled. During canonical (β-catenin–dependent) signaling, Dishevelled assembles signalosomes via dynamic head-to-tail polymerization of its Dishevelled and Axin (DIX) domain, which are cross-linked by its Dishevelled, Egl-10, and Pleckstrin (DEP) domain through a conformational switch from monomer to domain-swapped dimer. The domain-swapped conformation of DEP masks the site through which Dishevelled binds to Frizzled, implying that DEP domain swapping results in the detachment of Dishevelled from Frizzled. This would be incompatible with noncanonical Wnt signaling, which relies on long-term association between Dishevelled and Frizzled. It is therefore likely that DEP domain swapping is differentially regulated during canonical and noncanonical Wnt signaling. Here, we use NMR spectroscopy and cell-based assays to uncover intermolecular contacts in the DEP dimer that are essential for its stability and for Dishevelled function in relaying canonical Wnt signals. These contacts are mediated by an intrinsically structured sequence spanning a conserved phosphorylation site upstream of the DEP domain that serves to clamp down the swapped N-terminal α-helix onto the structural core of a reciprocal DEP molecule in the domain-swapped configuration. Mutations of this phosphorylation site and its cognate surface on the reciprocal DEP core attenuate DEP-dependent dimerization of Dishevelled and its canonical signaling activity in cells without impeding its binding to Frizzled. We propose that phosphorylation of this crucial residue could be employed to switch off canonical Wnt signaling.

Wnt 信号与 Frizzled 受体结合，触发在动物发育和组织稳态过程中控制细胞命运的典型和非典型信号反应。所有 Wnt 信号都由中枢蛋白 Dishevelled 中继。在典型（β-连环蛋白依赖性）信号传导过程中，Disheveled 通过其 Dishevelled 和 Axin (DIX) 结构域的动态头尾聚合组装信号体，这些结构域由其 Dishevelled、Egl-10 和 Pleckstrin (DEP) 交联通过从单体到结构域交换二聚体的构象转换。DEP 的域交换构象掩盖了 Disheveled 与 Frizzled 结合的位点，这意味着 DEP 域交换导致 Disheveled 从 Frizzled 分离。这与非规范的 Wnt 信号不兼容，这依赖于 Disheveled 和 Frizzled 之间的长期关联。因此，DEP 域交换很可能在规范和非规范 Wnt 信号传导期间受到差异调节。在这里，我们使用核磁共振波谱和基于细胞的分析来揭示 DEP 二聚体中的分子间接触，这对于其稳定性和传递典型 Wnt 信号的散乱功能至关重要。这些接触由跨越 DEP 结构域上游保守磷酸化位点的固有结构序列介导，该序列用于将交换的 N 末端 α-螺旋固定到结构域交换配置中的互惠 DEP 分子的结构核心上。该磷酸化位点及其在相互 DEP 核心上的同源表面的突变会减弱 DISHEVELLED 的 DEP 依赖性二聚化及其在细胞中的典型信号传导活性，而不会阻碍其与 Frizzled 的结合。我们建议可以使用这一关键残基的磷酸化来关闭典型的 Wnt 信号。