Abstract
Canonical Wnt signaling is controlled intracellularly by the level of β-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates β-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling.
Highlights
Wnt signaling is a fundamental morphogenetic pathway of metazoans that is deployed in diverse settings throughout development to regulate processes such as cell fate specification, stem cell regeneration, and neuronal migration [1]
By screening a siRNA library targeting all human ubiquitin E3 ligases, we have identified RNF146 as a new positive regulator of Wnt signaling
RNF146 acts with tankyrase in the constitutive turnover of Axin proteins that maintains low levels of Axin and allows the Wnt receptor-coreceptor complex to initiate signaling by further repressing Axin’s function in degrading b-catenin
Summary
Wnt signaling is a fundamental morphogenetic pathway of metazoans that is deployed in diverse settings throughout development to regulate processes such as cell fate specification, stem cell regeneration, and neuronal migration [1]. Wnt signaling can become deregulated through multiple mechanisms to produce cancer or other diseases, colorectal cancer for which APC or b-catenin is mutated in approximately 95% of tumors [2]. Many mechanisms have evolved to control the level, activity, and subcellular localization of multiple Wnt pathway components [3]. The best characterized mode of Wnt signaling regulation is the degradation of b-catenin by a protein complex that includes Axin and APC. This complex mediates the phosphorylation of bcatenin by CK1 and GSK3, which signals b-catenin ubiquitylation by the SCFb-TrCP complex to target b-catenin to the proteasome for proteolysis
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