In vivo analysis in Drosophila reveals differential requirements of contact residues in Axin for interactions with GSK3β or β-catenin

Abstract

Proper regulation of the Wingless/Wnt signaling pathway is essential for normal development. The scaffolding protein Axin plays a key role in this process through interactions with Drosophila Shaggy and Armadillo. In the current studies, we used a yeast two-hybrid assay to identify ten amino acids in Axin that are critical for in vitro interaction with Shaggy and two for interaction with Armadillo. We then generated five Axin variants in which individual putative contact amino acids were mutated and compared their activity, as assayed by rescue of axin null mutant flies, to that of Axin lacking the entire Shaggy (AxinΔSgg) or Armadillo (AxinΔArm) binding domain. Although we expected these mutants to function identically to Axin in which the entire binding domain was deleted, we instead observed a spectrum of phenotypic rescue. Specifically, two point mutants within the Shaggy binding domain showed loss of activity similar to that of AxinΔSgg and dominantly interfered with complex function, whereas a third mutant allele, AxinK446E retained most function. Two Axin point mutants within the Armadillo binding domain were weak alleles, and retained most function. These findings demonstrate the importance of in vivo verification of the role of specific amino acids within a protein.

Identification of Proteins Required for Precise Positioning of Apc2 in Dendrites

“…However, the data strongly indicates that frizzleds, Gαo and Gαs act to position Apc2, and all except Gαs likely act through Axin as they are required for its positioning. The ability of activated and inactive forms of sgg (GSK3β) to modulate localization of Apc2-GFP and Axin-GFP are consistent with a subset of wnt signaling proteins playing a role in branch point localization as sgg can bind Axin ( kremer et al 2010 ) as well as other wnt signaling proteins. However, we do not have evidence that sgg is normally involved in branch point localization at this point because the RNAi that targets sgg had no effect in any of the assays.…”

An early global role for Axin is required for correct patterning of the anterior-posterior axis in the sea urchin embryo

“…For example, overexpression of Axin constructs lacking the GID in Xenopus failed to ventralize embryos, while a Drosophila Axin ΔGID protein expressed at near endogenous levels was unable to rescue embryos. Presumably, the loss of activity of these proteins was due to their inability to regulate the β-catenin destruction complex ( Fagotto et al, 1999 ; Hedgepeth et al, 1999 ; Itoh et al, 1998 ; Kremer et al, 2010 ; Peterson-Nedry et al, 2008 ). We showed that overexpression of Axin GID alone produced a phenotype reminiscent of those produced by downregulation of GSK-3β activity or overexpression of β-catenin ( Emily-Fenouil et al, 1998 ; Wikramanayake et al, 1998 ).…”

Wnt/Beta-Catenin Signaling Regulation and a Role for Biomolecular Condensates

“…This makes it surprisingly robust to removal of some but not all protein interaction motifs. For example, individually deleting most of APC's bCat-binding sites, Axin's bCat-binding site, or Axin's RGS domain have only modest effects in vivo (Kremer et al, 2010;Kunttas-Tatli et al, 2012;Peterson-Nedry et al, 2008;Roberts et al, 2011;Yamulla et al, 2014). However, some binding sites are essential individually (Axin's GSK-3-binding site) or when deleted in concert (AxinDRGSDArm; Peterson-Nedry et al, 2008).…”