👤 Ondrej Bernatik

Primary cilia act as crucial regulators of embryo development and tissue homeostasis. They are instrumental for modulation of several signaling pathways, including Hedgehog, WNT, and TGF-β. However, gaps exist in our understanding of how cilia formation and function is regulated. Recent work has implicated WNT/β-catenin signaling pathway in the regulation of ciliogenesis, yet the results are conflicting. One model suggests that WNT/β-catenin signaling negatively regulates cilia formation, possibly via effects on cell cycle. In contrast, second model proposes a positive role of WNT/β-catenin signaling on cilia formation, mediated by the re-arrangement of centriolar satellites in response to phosphorylation of the key component of WNT/β-catenin pathway, β-catenin. To clarify these discrepancies, we investigated possible regulation of primary cilia by the WNT/β-catenin pathway in cell lines (RPE-1, NIH3T3, and HEK293) commonly used to study ciliogenesis. We used WNT3a to activate or LGK974 to block the pathway, and examined initiation of ciliogenesis, cilium length, and percentage of ciliated cells. We show that the treatment by WNT3a has no- or lesser inhibitory effect on cilia formation. Importantly, the inhibition of secretion of endogenous WNT ligands using LGK974 blocks WNT signaling but does not affect ciliogenesis. Finally, using knock-out cells for key WNT pathway components, namely DVL1/2/3, LRP5/6, or AXIN1/2 we show that neither activation nor deactivation of the WNT/β-catenin pathway affects the process of ciliogenesis. These results suggest that WNT/β-catenin-mediated signaling is not generally required for efficient cilia formation. In fact, activation of the WNT/β-catenin pathway in some systems seems to moderately suppress ciliogenesis. Show less

Dishevelled (Dvl) is a key component in the Wnt/β-catenin signaling pathway. Dvl can multimerize to form dynamic protein aggregates, which are required for the activation of downstream signaling. Upon pathway activation by Wnts, Dvl becomes phosphorylated to yield phosphorylated and shifted (PS) Dvl. Both activation of Dvl in Wnt/β-catenin signaling and Wnt-induced PS-Dvl formation are dependent on casein kinase 1 (CK1) δ/ε activity. However, the overexpression of CK1 was shown to dissolve Dvl aggregates, and endogenous PS-Dvl forms irrespective of whether or not the activating Wnt triggers the Wnt/β-catenin pathway. Using a combination of gain-of-function, loss-of-function, and domain mapping approaches, we attempted to solve this discrepancy regarding the role of CK1ε in Dvl biology. We analyzed mutual interaction of CK1δ/ε and two other Dvl kinases, CK2 and PAR1, in the Wnt/β-catenin pathway. We show that CK2 acts as a constitutive kinase whose activity is required for the further action of CK1ε. Furthermore, we demonstrate that the two consequences of CK1ε phosphorylation are separated both spatially and functionally; first, CK1ε-mediated induction of TCF/LEF-driven transcription (associated with dynamic recruitment of Axin1) is mediated via a PDZ-proline-rich region of Dvl. Second, CK1ε-mediated formation of PS-Dvl is mediated by the Dvl3 C terminus. Furthermore, we demonstrate with several methods that PS-Dvl has decreased ability to polymerize with other Dvls and could, thus, act as the inactive signaling intermediate. We propose a multistep and multikinase model for Dvl activation in the Wnt/β-catenin pathway that uncovers a built-in de-activation mechanism that is triggered by activating phosphorylation of Dvl by CK1δ/ε. Show less