Functional dissection of phosphorylation of Disheveled in Drosophila

Abstract

Dishevelled/Dsh proteins (Dvl in mammals) are core components of both Wnt/Wg-signaling pathways: canonical β-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 - DshY473F behaves like a PCP-specific allele; and (3) defects associated with the PCP specific dsh1 allele, DshK417M, 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.

Atypical Protein Kinase Cι Is Required for Wnt3a-dependent Neurite Outgrowth and Binds to Phosphorylated Dishevelled 2

“…A recent extensive analysis identified dozens of phosphorylated Ser/Thr residues in Dvl (or the Drosophila ortholog, Dsh). However, these modifications were dispensable for ␤-catenin and planar cell polarity signaling in the Drosophila assays (38). The present study and our accompanying report (43) suggest that Dvl2 phosphorylation, particularly at Ser 594 , Thr 595 , and Ser 597 , is relevant for signaling in a Wnt/aPKC pathway.…”

The E3 Ubiquitin Ligase ITCH Negatively Regulates Canonical Wnt Signaling by Targeting Dishevelled Protein

“…Similarly, ITCH ubiquitinates and promotes the degradation of only phosphorylated Dvl, which is different from the alreadyfound E3 ubiquitin ligases for Dvl, such as the KLHL12-Cullin-3 complex, pVHL, NEDL1, and Malin (1,10,26,37), whose ubiquitination of Dvl does not relate to Dvl's phosphorylation state. A previous study reported that Dsh (the Dvl homolog in flies) phosphorylation does not seem to have a major effect on canonical Wnt signaling in Drosophila (43). This may reflect intrinsic differences in the fine regulation of the Wnt signaling pathways between the vertebrate and invertebrate systems.…”

Functional Consequences of Wnt-induced Dishevelled 2 Phosphorylation in Canonical and Noncanonical Wnt Signaling

“…3). Dozens of phosphorylation sites have been documented in Dvl proteins, and some have been identified as targets of specific kinases (35)(36)(37)(38). Although phosphorylation sites are widely distributed in the Dvl sequence, they are particularly abundant in the CTD (35).…”

“…Dozens of phosphorylation sites have been documented in Dvl proteins, and some have been identified as targets of specific kinases (35)(36)(37)(38). Although phosphorylation sites are widely distributed in the Dvl sequence, they are particularly abundant in the CTD (35). Deletion analysis and site-directed mutagenesis have suggested that phosphorylation in this region correlates with formation of psDvl (12,35,39).…”