Abstract
Feedback control is a universal feature of cell signaling pathways. Naked/NKD is a widely conserved feedback regulator of Wnt signaling which controls animal development and tissue homeostasis. Naked/NKD destabilizes Dishevelled, which assembles Wnt signalosomes to inhibit the β-catenin destruction complex via recruitment of Axin. Here, we discover that the molecular mechanism underlying Naked/NKD function relies on its assembly into ultra-stable decameric core aggregates via its conserved C-terminal histidine cluster (HisC). HisC aggregation is facilitated by Dishevelled and depends on accumulation of Naked/NKD during prolonged Wnt stimulation. Naked/NKD HisC cores co-aggregate with a conserved histidine cluster within Axin, to destabilize it along with Dishevelled, possibly via the autophagy receptor p62, which binds to HisC aggregates. Consistent with this, attenuated Wnt responses are observed in CRISPR-engineered flies and human epithelial cells whose Naked/NKD HisC has been deleted. Thus, HisC aggregation by Naked/NKD provides context-dependent feedback control of prolonged Wnt responses.
Highlights
Cells communicate via a handful of highly conserved signaling pathways to coordinate growth and differentiation
The Nkd1 histidine cluster (HisC) is crucial for ternary complex formation with Axin and DVL2 We used co-overexpression assays in HEK293T cells, monitoring Wnt signaling with a cotransfected β-catenin-dependent transcriptional reporter (SuperTOP) (Veeman et al, 2003), to confirm that murine HA-Nkd1 reduces the signaling activity of co-overexpressed DVL2-GFP, but not of co-overexpressed β-catenin (Fig. S2)
While conducting these coIP assays, we discovered that overexpressed Nkd1 forms high-molecular weight (HMW) aggregates that are resistant to boiling in sodium dodecyl sulphate (SDS), and whose formation critically depends on HisC (Fig. 1C)
Summary
Cells communicate via a handful of highly conserved signaling pathways to coordinate growth and differentiation. Dishevelled transduces Wnt signals through canonical or non-canonical effector branches to elicit distinct cellular readouts, whereby the best-studied one is the β-catenin-dependent canonical branch which requires interaction of Dishevelled with Axin (Angers and Moon, 2009; Gammons and Bienz, 2018). In order to transduce Wnt signals to β-catenin, Dishevelled assembles signalosomes by dynamic head-to-tail polymerization of its DIX domain (Schwarz-Romond et al, 2007). This enables it to bind to the DIX domain of Axin, which leads to inhibition of the associated kinases in the β-catenin destruction complex (Stamos et al, 2014) whose assembly, in the absence of signaling, depends on polymerization by the Axin DIX domain (Fiedler et al, 2011). The flow through the canonical Wnt signaling pathway is determined by the opposing activities of polymerizing Axin and Dishevelled and their mutual interaction via their DIX domains (Bienz, 2014): the polymerization of Axin promotes destabilization of β-catenin and ensures quiescence of the pathway, while the polymerization of Dishevelled allows signalosome assembly and co-polymerization with Axin, which allows β-catenin to accumulate in the nucleus and engage in transcriptional activation of Wnt target genes (Gammons and Bienz, 2018)
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