Ubiquitin Ligase RNF146 Regulates Tankyrase and Axin to Promote Wnt Signaling

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.

Regulation of NF-κB signalling by the mono-ADP-ribosyltransferase ARTD10

“…Recent studies have demonstrated that ARTD5-and 6-(formerly Tankyrase1 and 2) dependent PARylation of axin results in the recruitment of the ubiquitin E3 ligase RNF146 and subsequent pUb-dependent proteasomal degradation of axin, a negative regulator of WNT signalling 9,10,25 . Similarly, 3BP2 is substrate of ARTD5 and RNF146.…”

“…PARylation regulates various cellular processes, such as DNA repair, specific signalling processes and gene transcription 5 . The functional consequences of PARylation are mediated by distinct protein domains, including macrodomains, WWE (named after two conserved tryptophan residues and a glutamate residue) and PAR-binding zinc-finger (PBZ) domains [6][7][8][9][10] . In contrast to the function of class 1 ARTD enzyme-mediated PARylation, little is known about the biological relevance of mono-ADP-ribosyltransferases.…”

HectD1 E3 Ligase Modifies Adenomatous Polyposis Coli (APC) with Polyubiquitin to Promote the APC-Axin Interaction

“…FuGENE 6 HD (Roche Applied Science) was used for plasmid transfection in RPE cells. Epitope-tagged Trabid, HectD1, APC, Tankyrase (TNKS1) and RNF146 constructs have been described (17,24,25). Recombinant mouse Wnt3a was purchased from R & D Systems.…”

“…This protein was incubated with lysates from HEK293 cells expressing HA-HectD1. To control for binding specificity, we used a Glu-Glu-tagged APC3 protein (gg-APC3) purified from insect cells (27) and lysates from HEK293 cells expressing HATankyrase (TNKS1), a poly(ADP-ribosyl)ase that binds the E3 ligase RNF146 to cooperatively down-regulate Axin (17). Following pulldown with anti-Glu-Glu-agarose, we detected an interaction between HA-HectD1 and the APC ARD, but only weak association between HA-HectD1 and APC3 (Fig.…”

“…The stabilities of Axin and Dishevelled, an upstream positive component of the pathway, are regulated by the E3 ligase activities of RNF146 and KLHL12, respectively. The KLHL12-mediated proteasomal degradation of Dishevelled is a Wnt-regulated event, but it is unclear whether the same is true of RNF146-mediated Axin degradation (15)(16)(17). Like KLHL12, the recently described ZNRF3 represents another E3 ubiquitin ligase that negatively regulates proximal events in the Wnt pathway.…”