Untitled

Abstract

The DEP domain of Dishevelled (Dvl) proteins transduces signals to effector proteins downstream of Dvl in the Wnt pathway. Here we report that DEP-containing mutants inhibit Wnt-induced, but not Dvl-induced, activation of the transcription factor Lef-1. This inhibitory effect is weakened by a K434M mutation. Nuclear magnetic resonance spectroscopy revealed that the DEP domain of mouse Dvl1 comprises a three-helix bundle, a β-hairpin 'arm' and two short β-strands at the Cterminal region. Lys 434 is located at the tip of the β-hairpin 'arm'. Based on our findings, we conclude that DEP interacts with regulators upstream of Dvl via a strong electric dipole on the molecule's surface created by Lys 434, Asp 445 and Asp 448; the electric dipole and the putative membrane binding site are at two different locations.The Wnt signaling pathway is a key regulatory pathway for cellular development and growth. Wnt signaling has been extensively studied in Drosophila, Caenorhabditis elegans, Xenopus, and mammalian systems [1][2][3][4][5] . Through genetic studies, a working model of the Wnt signaling pathway has been established. Secreted Wnt proteins bind to Frizzled transmembrane receptors. The receptor-ligand complex activates the Dishevelled (Dsh and Dvl) proteins, which suppress the activity of glycogen synthase kinase-3β (GSK-3β). If its activity is not suppressed, GSK-3β binds to adenomatous polyposis coli (APC) protein and axin and then phosphorylates specific Ser and Thr residues at the N-terminus of β-catenin. In turn, the ubiquitin-proteasome pathway rapidly degrades hyperphosphorylated β-catenin. Wnt activation prevents this process by promoting the cytosolic accumulation and subsequent translocation of β-catenin into the nucleus. Once in the nucleus, β-catenin binds to the transcription factors T-cell factor (Tcf) or lymphoid enhancer factor (Lef) and acts as a transcriptional coactivator 5 . The result is increased expression of Tcf or Lef regulated target genes, such as Myc 6 and the gene encoding cyclin D 7 .© 2000 Nature America Inc. Correspondence should be addressed to: J.Z. Jie.Zheng@stjude.org. HHS Public Access DEP domain interacts with upstream regulators of DvlWe and others have demonstrated that Wnt-1 or Dvl1, when expressed in NIH3T3 cells, can activate the transcription regulator 16,28,29). This activation depends on β-catenin and is suppressed by GSK-3β and axin. To characterize the role of the mDvl1 DEP domain in Wnt signaling, we examined the effect of the Dvl1 N-terminal truncation mutant, DvlC1, on Wnt-induced activation of the transcription factor Lef-1. In Lef dependent reporter gene assays, DvlC1 inhibited Wnt-1 induced Lef-1 activation but not Dvl1 induced Lef-1 activation (Fig. 2). To further characterize the specific sequences that inhibit the Wnt-induced activation, we generated two additional mutants of mDvl1: DvlDEP, which contains only the DEP domain; and DvlC2, which encompasses the sequence downstream of the DEP domain. When expressed in NIH3T3 cells, DvlDEP, like DvlC1, bloc...

A disorder-to-order structural transition in the COOH-tail of Fz4 determines misfolding of the L501fsX533-Fz4 mutant

“…2b). The existence of this short helix has already been postulated and shown to be involved in the interaction of Fz4 with the effector protein Dishevelled1527, while the remaining part of the wt tail (from Leu 511 to Val 537 ) was predicted to be disordered27. We judged the structure prediction for the mutant tail sound and worthwhile of further experimental proof.…”

“…Two PDZ binding motives are located one internally and one at the C-terminus of Fz4. The cytosolic tail of Fz4 has been shown to interact with the protein Dishevelled to activate several signalling pathways141516. The frameshift mutation L501fsX533 of Fz4 is associated with a rare form of Familial exudative vitreorethinopaty (FEVR)17 that shows an autosomal dominant inheritance.…”

Functional dissection of phosphorylation of Disheveled in Drosophila

“…However, our data indicate that it is the Lysine itself that is essential and a mutation to the related Arginine residue (which is similarly charged and often can substitute for Lys) also causes the same defects as the original DshK417M mutation. These data suggest that it is a post-translational modification such as ubiquitination or acetylation of Lys417, as it does not appear to be of structural importance (Wong et al, 2000). This notion is consistent with recent work in mammalian cell culture (Ganner et al, 2009), although we cannot exclude a direct involvement of Lys417.…”

Genetic Analysis of Disheveled 2 and Disheveled 3 in Human Neural Tube Defects

“…They function as essential scaffold proteins that interact with diverse proteins, including kinases, phosphatases, and adaptor proteins. They contain three highly conserved domains: the DIX (Disheveled/Axin) domain that is largely α-helical structure (Capelluto et al 2002), the PDZ (PSD-95, DLG,ZO1) domain, which consists of six β-sheets that enfold two α-helices (Wong et al 2003), and the DEP (Disheveled, EGL-10, Pleckstrin) domain, consisting of a bundle of three α-helices domain (Wong et al 2000). Several additional conserved regions are considered critical for biological function: the serine/threonine stretches between the DIX and PDZ domains, and the proline-rich regions with a SH3 protein-binding domain motif downstream of the PDZ.…”