Investigating the Role of Casein Kinase 1ε Phosphorylation of Dvl3 in Mammalian Planar Cell Polarity

Abstract

Planar cell polarity (PCP) is an essential pathway in mammalian embryonic development by which cells across various tissues establish and propagate an axis of polarity necessary for coordinated cell behaviors. Loss of this planar polarization induces severe congenital diseases in humans, such as spina bifida, hearing deficits, and neural tube defects, among many others. A critical requirement of PCP is the asymmetric localization of its highly conserved core PCP proteins. Among these proteins, cytoplasmic Dishevelled (Dvl) is an essential PCP protein with roles in propagating planar polarization across the tissue sheet. A key component of PCP signaling on the cellular level is the shift of Dvl localization from the cytoplasm, where it can exist uniformly or in phase-separated condensates, to cell-cell junctions, where it associates with the transmembrane protein Frizzled (Fz). Despite the importance of Dvl relocalization during PCP, there is little mechanistic insight into how Dvl associates at cell-cell junctions. Important features of the Dvl protein is that it is heavily phosphorylated with over 50 Casein Kinase 1ε (CK1ε) phosphorylation sites identified as well as an exceptional ability to phase separate into puncta, which may be a potential mechanism by which Dvl associates with Fz at high density at cell-cell junctions. In this study, I characterize the role of CK1ε phosphorylation on the ability for Dvl3 to phase separate and localize at junctions using mouse keratinocytes derived from the epidermis as a model system of PCP. Mutagenesis analyses of Dvl3 showed that the phosphorylation of Dvl3 at CK1ε phosphorylation sites S280 and S311 enhanced junctional recruitment but attenuated phase separation into puncta. Further, pharmacological inhibition of the CK1ε kinase on stage E15.5 mouse epidermal skin explants demonstrated that CK1ε is required for proper hair follicle polarization as normal follicle alignment was lost after inhibitor treatment. This unexpected finding revealed more broadly that PCP functioning may not be restrained within a precise developmental time window as commonly described in scientific literature. PCP may rather be a process that must be maintained through key events, including the CK1ε phosphorylation of Dvl3. Further study of the condensation of Dvl3 puncta and its interaction with the cytoplasmic domain of Fz through the development of assays using purified proteins on artificial membranes could provide deeper insights about the role of phase separation in the context of the plasma membrane.