Armed with PRICKLE(3)s: Stabilizing WNT/PCP complexes against RNF43-mediated ubiquitination

The human Prickle protein family, consisting of PRICKLE1, PRICKLE2, PRICKLE3, and PRICKLE4, is an integral component of the WNT/planar cell polarity (WNT/PCP) pathway and is essential for various cellular and developmental processes. Despite their significance, the detailed roles and involvement in molecular mechanisms of these proteins in cells remain not fully understood. In this study, we used enhanced proximity biotinylation (miniTurboID) combined with mass spectrometry to characterize the microenvironment of PRICKLE1-3. Our results reveal that PRICKLE3 is directly linked to the WNT/PCP pathway, primarily localizing at the plasma membrane and forming complexes with VANGL proteins. This observation prompted us to examine its role in the non-canonical WNT signalling pathway in more detail. Using an inducible expression system to achieve protein levels closer to physiological conditions, we found that PRICKLE3 enhances the stability of VANGL1 and VANGL2 by shielding them from Casein kinase 1 {varepsilon}-mediated phosphorylation. Furthermore, our results indicate that PRICKLE3 modulates WNT receptor complexes by negatively regulating the interaction between Casein kinase 1 {varepsilon} and ubiquitin ligase RNF43, resulting in decreased ubiquitination and increased stabilization of VANGL1/2 at the plasma membrane. Notably, these effects were specific to PRICKLE3, with PRICKLE1 showing no comparable activity. Contrary to previous findings based mainly on standard overexpression studies, neither PRICKLE3 nor PRICKLE1 influenced the levels or phosphorylation status of WNT proteins DISHEVELLED2 and DISHEVELLED3, which are the PRICKLE proteins binding partners. In summary, we have identified a key mechanism specific to PRICKLE3 that positively regulates WNT/PCP complexes by suppressing RNF43. Additionally, we present a comprehensive interactome and new tools for the functional specification of Prickle isoforms to support further research.