Abelson family kinases regulate Frizzled planar cell polarity signaling via Dsh phosphorylation.

Abelson (Abl) family tyrosine kinases have been implicated in cell morphogenesis, adhesion, motility, and oncogenesis. Using a candidate approach for genes involved in planar cell polarity (PCP) signaling, we identified Drosophila Abl (dAbl) as a modulator of Frizzled(Fz)/PCP signaling. We demonstrate that dAbl positively regulates the Fz/Dishevelled (Dsh) PCP pathway without affecting canonical Wnt/Wg-Fz signaling. Genetic dissection suggests that Abl functions via Fz/Dsh signaling in photoreceptor R3 specification, a well-established Fz-PCP signaling readout. Molecular analysis shows that dAbl binds and phosphorylates Dsh on Tyr473 within the DEP domain. This phosphorylation event on Dsh is functionally critical, as the equivalent DshY473F mutant is nonfunctional in PCP signaling and stable membrane association, although it rescues canonical Wnt signaling. Strikingly, mouse embryonic fibroblasts (MEFs) deficient for Abl1 and Abl2/Arg genes also show reduced Dvl2 phosphorylation as compared with control MEFs, and this correlates with a change in subcellular localization of endogenous Dvl2. As in Drosophila, such Abl-deficient MEFs show no change in canonical Wnt signaling. Taken together, our results argue for a conserved role of Abl family members in the positive regulation of Dsh activity toward Fz-Dsh/PCP signaling by Dsh phosphorylation.

Functional dissection of phosphorylation of Disheveled in Drosophila.

Disheveled/Dsh proteins (Dvl in mammals) are core components of both Wnt/Wg-signaling pathways: canonical ß-catenin signaling and Frizzled (Fz)-planar cell polarity (PCP) signaling. Although Dsh is a key cytoplasmic component of both Wnt/Fz-pathways, regulation of its signaling specificity is not well understood. Dsh is phosphorylated, but the functional significance of its phosphorylation remains unclear. We have systematically investigated the phosphorylation of Dsh by combining mass-spectrometry analyses, biochemical studies, and in vivo genetic methods in Drosophila. Our approaches identified multiple phospho-residues of Dsh in vivo. Our data define three novel and unexpected conclusions: (1) strikingly and in contrast to common assumptions, all conserved serines/threonines are non-essential for Dsh function in either pathway; (2) phosphorylation of conserved Tyrosine473 in the DEP domain is critical for PCP-signaling - Dsh(Y473F) behaves like a PCP-specific allele; and (3) defects associated with the PCP specific dsh(1) allele, Dsh(K417M), located within a putative Protein Kinase C consensus site, are likely due to a post-translational modification requirement of Lys417, rather than phosphorylation nearby. In summary, our combined data indicate that while many Ser/Thr and Tyr residues are indeed phosphorylated in vivo, strikingly most of these phosphorylation events are not critical for Dsh function with the exception of DshY473.

Frizzled-Induced Van Gogh Phosphorylation by CK1ε Promotes Asymmetric Localization of Core PCP Factors in Drosophila.

Epithelial tissues are polarized along two axes. In addition to apical-basal polarity, they are often polarized within the plane of the epithelium, so-called Planar Cell Polarity (PCP). PCP depends upon Wnt/Frizzled (Fz) signaling factors, including Fz itself and Van Gogh (Vang/Vangl). We sought to understand how Vang interaction with other core PCP factors affects Vang function. We find that Fz induces Vang phosphorylation in a cell-autonomous manner. Vang phosphorylation occurs on conserved N-terminal serine/threonine residues, is mediated by CK1ε/Dco, and is critical for polarized membrane localization of Vang and other PCP proteins. This regulatory mechanism does not require Fz signaling through Dishevelled and thus represents a cell-autonomous upstream interaction between Fz and Vang. Furthermore, this signaling event appears to be related to Wnt5a-mediated Vangl2 phosphorylation during mouse limb patterning and may thus be a general mechanism underlying Wnt-regulated PCP establishment.

Phosphorylation of frizzled-3.

Wnts are secreted proteins important to many biological processes. frizzled genes encode a family of Wnt receptors that signal to the intracellular compartment through the cytosolic protein Disheveled. Limited information is available concerning the regulation of Frizzleds at a biochemical level. We report here that Xenopus Frizzled-3 is phosphorylated in a Disheveled-dependent manner that appears to require the DEP domain of Disheveled. Phosphorylation of serine 576 causes a decrease in electrophoretic mobility and accounts for a significant fraction of receptor phosphorylation, although additional residues in the C-terminal tail are also phosphorylated. In addition, mutations that interfere with Frizzled-3 function also interfere with phosphorylation, but these inactive mutants can be phosphorylated when an active form of Frizzled-3 is co-expressed. Mutation of C-terminal serines including serine 576 significantly enhances Frizzled-3-mediated induction of neural crest markers, suggesting that C-terminal phosphorylation plays a role in down-regulating Frizzled signaling.