Characterization of the signaling mechanisms of core Planar cell polarity (PCP) protein Vangl2

Abstract

Planar cell polarity (PCP) refers to the coordinated orientation of a group of cells within the plane of a cell sheet in tissue morphogenesis and organogenesis. Disruption of PCP results in a variety of human diseases, such as neural tube defects, neurological disorders, skeletal dysplasias, and cancers. PCP is controlled by a set of core proteins (e.g., Fzd, Dvl, Vangl, Pk and Celsr), which are segregated to the opposite sides of the cell in the polarized tissues. However, the downstream signaling mechanisms of these core PCP proteins remain elusive. In this study, I first utilized the BioID-based proximity-dependent biotinylation approach to identify interactors of Vangl2, which led to the identification of more than 300 proteins. I characterized 10 candidates and 6 of them showed positive interaction with Vangl2. I then studied a serine/threonine kinase Mark3 that strongly interacted with Vangl2 in the presence of Dvl and, based on in silico analysis, Mark3 potentially phosphorylates Vangl2 at S517. However, the following in vitro kinase assay and cell-based assays revealed that PKD1 and Akt1 rather than Mark3 were the responsible kinases for S517 phosphorylation of Vangl2. Wnt and EGF ligands could stimulate the phosphorylation of S517 through PKD1 and Akt1, respectively. The phosphorylation of S517 was not essential for Vangl2 stability and membrane localization, but intriguingly, S517 phosphorylation is required for the binding of Vangl2 to Scrib, a key apical-basal (A/B) polarity protein. Such interaction has been shown to be important for the crosstalk between the PCP and A/B polarity. I found that S517 phosphorylation of Vangl2 regulated the convergent extension process in zebrafish gastrulation and synaptogenesis in cultured neurons. To better understand the function of S517 phosphorylation in vivo, I further generated a Vangl2 knock-in mouse model carrying the S517A mutation. In the Vangl2 S517A/S517A mutant, the abolishment of S517 phosphorylation indeed significantly disrupted the binding between Vangl2 and Scrib but no overt PCP phenotype was observed. The mouse behavioral tests also showed that the anxiety-related behavior and learning and memory ability were not significantly affected in the mutant mice. In summary, my studies uncovered a new phosphorylation-mediated signaling mechanism that is important for the interaction of the core PCP protein Vangl2 with the key A/B polarity protein Scrib, but surprisingly, its in vivo function remains obscure, which requires further studies both genetically and biochemically.