The canonical Wnt signaling pathway plays a crucial role in embryonic development, tissue homeostasis and cancer progression. The binding of Wnt ligands to their cognate receptors, the Frizzled (Fzd) family of proteins, recruits Dishevelled segment polarity protein (Dvl) to the plasma membrane and induces its phosphorylation via casein kinase 1 (CK1), which leads to the activation of beta-catenin. Previous studies showed that Dishevelled-associating protein with a high frequency of leucine residues (Daple) is an important component of the Wnt signaling pathway and essential for Dvl phosphorylation. However, the mechanism by which Daple promotes CK1-mediated phosphorylation of Dvl is not fully understood. In this study, we found that Daple overexpression induced CK1 epsilon-mediated Dvl2 phosphorylation at threonine 224 (Thr224). A Daple mutant (Daple Delta GCV) that lacks a carboxyl-terminal motif to associate with Dvl, retained the ability to interact with CK1 epsilon, but did not induce Dvl phosphorylation, suggesting the importance of the Daple/Dvl/CK1 epsilon trimeric protein complex. We further found that Thr224 phosphorylation of Dvl was required for full activation of beta-catenin transcriptional activity. Consistent with this, wild-type Daple promoted beta-catenin transcriptional activity, following dissociation of beta-catenin and axin. Finally, Wnt3a stimulation increased the membrane localization of Daple and its association with Dvl, and Daple knockdown attenuated Wnt3a-mediated beta-catenin transcriptional activity. Collectively, these data suggested a essential role of spatial Daple localization in CK1 epsilon-mediated activation of Dvl in the canonical Wnt signaling pathway. (C) 2020 Elsevier Inc. All rights reserved.