Dual Roles for Membrane Association of Drosophila Axin in Wnt Signaling

Abstract

Deregulation of the Wnt signal transduction pathway underlies numerous congenital disorders and cancers. Axin, a concentration-limiting scaffold protein, facilitates assembly of a “destruction complex” that prevents signaling in the unstimulated state and a plasma membrane-associated “signalosome” that activates signaling following Wnt stimulation. In the classical model, Axin is cytoplasmic under basal conditions, but relocates to the cell membrane after Wnt exposure; however, due to the very low levels of endogenous Axin, this model is based largely on examination of Axin at supraphysiological levels. Here, we analyze the subcellular distribution of endogenous Drosophila Axin in vivo and find that a pool of Axin localizes to cell membrane proximal puncta even in the absence of Wnt stimulation. Axin localization in these puncta is dependent on the destruction complex component Adenomatous polyposis coli (Apc). In the unstimulated state, the membrane association of Axin increases its Tankyrase-dependent ADP-ribosylation and consequent proteasomal degradation to control its basal levels. Furthermore, Wnt stimulation does not result in a bulk redistribution of Axin from cytoplasmic to membrane pools, but causes an initial increase of Axin in both of these pools, with concomitant changes in two post-translational modifications, followed by Axin proteolysis hours later. Finally, the ADP-ribosylated Axin that increases rapidly following Wnt stimulation is membrane associated. We conclude that even in the unstimulated state, a pool of Axin forms membrane-proximal puncta that are dependent on Apc, and that membrane association regulates both Axin levels and Axin’s role in the rapid activation of signaling that follows Wnt exposure.

Regulation of Wnt/β‐catenin signalling by tankyrase‐dependent poly(ADP‐ribosyl)ation and scaffolding

“…Interestingly, while PARP activity is dispensable for tankyrase‐driven Wnt/β‐catenin signalling under basal conditions (Mariotti et al, ), it is necessary for tankyrase to potentiate Wnt‐induced β‐catenin activity (Riccio et al, ). This may well reflect the recently discovered requirement of AXIN PARylation in the formation of Wnt‐induced signalosomes (see below) (Yang et al, ; Wang et al, ) (Figure B).…”

“…A tankyrase‐associated ubiquitin‐specific protease (USP25) can de‐ubiquitylate tankyrase, thereby stabilizing it and supporting PARdU of AXIN (Xu et al, ). Recent studies point toward another role of tankyrase, namely in promoting the formation of active, membrane‐localized Wnt signalosomes upon Wnt stimulation (Yang et al, ; Wang et al, ) (Figure B). Furthermore, additional tankyrase interactors in the Wnt/β‐catenin pathway, other than AXIN, are emerging (Croy et al, ).…”

“…While tankyrase's role in PARdU of AXIN is well established, two recent studies point to a novel function in Wnt signalosome assembly (Yang et al, ; Wang et al, ) (Figure B). During Drosophila embryonic development, the abundance of weakly expressed Axin‐V5 changes in a biphasic manner (Yang et al, ).…”

“…Capitalizing on the ability to detect endogenous Drosophila Axin, a second study suggests that Axin is present in both the cytoplasm and at the membrane, even under basal conditions. Wg stimulation results in Axin accumulation in both compartments, with an enrichment of PARylated Axin at the membrane (Wang et al, ). Forcing Axin to the membrane under basal conditions increases its PARylation and destabilization, presumably by PARdU, suggesting that tankyrase acts at the membrane or Axin's susceptibility to PARylation is augmented upon membrane recruitment (Wang et al, ).…”

“…Wg stimulation results in Axin accumulation in both compartments, with an enrichment of PARylated Axin at the membrane (Wang et al, ). Forcing Axin to the membrane under basal conditions increases its PARylation and destabilization, presumably by PARdU, suggesting that tankyrase acts at the membrane or Axin's susceptibility to PARylation is augmented upon membrane recruitment (Wang et al, ). Phosphorylation by GSK3 (Kim et al, ) or binding of the small molecule HLY78 (Wang et al, ) was reported to induce an open AXIN conformation for productive interaction with LRP6.…”