Ubiquitin Ligase RNF146 Regulates Tankyrase and Axin to Promote Wnt Signaling

University of Birmingham, United Kingdom
PLoS ONE (Impact Factor: 3.23). 07/2011; 6(7):e22595. DOI: 10.1371/journal.pone.0022595
Source: PubMed


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.

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    • "Given that structural and biochemical analyses revealed that the binding specificity of these domains are dependent on the neighboring amino acids surrounding the modified sites [62] [68], these macrodomains will likely enrich for a restricted set of native ADP-ribosylated partners within cells. Besides macrodomains, the WWE domain from RNF146 has also been shown to be responsible for binding PARylated PARP1 and PARP5a [69] [70] [71]. Given that the WWE domain specifically recognizes the smallest structural subunit for PAR, iso-ADP-ribose [72] (c.f. "
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    ABSTRACT: ADP-ribosylation refers to the addition of one or more ADP-ribose units onto protein substrates and this protein modification has been implicated in various cellular processes including DNA damage repair, RNA metabolism, transcription and cell cycle regulation. This review focuses on a compilation of large-scale proteomics studies that identify ADP-ribosylated proteins and their associated proteins by mass spectrometry using a variety of enrichment strategies. Some methods, such as the use of a poly(ADP-ribose)-specific antibody and boronate affinity chromatography and NAD+ analogues, have been employed for decades while others, such as the use of protein microarrays and recombinant proteins that bind ADP-ribose moieties (such as macrodomains), have only recently been developed. The advantages and disadvantages of each method and whether these methods are specific for identifying mono(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins will be discussed. Lastly, since poly(ADP-ribose) is heterogeneous in length, it has been difficult to attain a mass signature associated with the modification sites. Several strategies on how to reduce polymer chain length heterogeneity for site identification will be reviewed.This article is protected by copyright. All rights reserved
    Proteomics 09/2014; 15(2-3). DOI:10.1002/pmic.201400217 · 3.81 Impact Factor
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    • "β-catenin associates with the DC, is phosphorylated by CK1-α and GSK3β 10–12, and subsequently ubiquitinated and degraded 13,14. Recently, it was shown that TNKS, at least in part, regulates this process through poly (ADP ribosyl)ating AXIN and itself, as well as the ubiquitin ligase RNF146, a process that initiates ubiquitination and degradation 15–18. Thus, through the control of the stability of the rate-limiting DC protein AXIN1/2, β-catenin levels can be attenuated by TNKS 19. "
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    ABSTRACT: Wnt/β-catenin is a major regulator of stem cell self-renewal and differentiation and this signaling pathway is aberrantly activated in a several cancers, including osteosarcoma (OS). Attenuation of Wnt/β-catenin activity by tankyrase inhibitors is an appealing strategy in treatment of OS. The efficacy of the tankyrase inhibitor JW74 was evaluated in three OS cell lines (KPD, U2OS, and SaOS-2) both at the molecular and functional level. At the molecular level, JW74 induces stabilization of AXIN2, a key component of the β-catenin destruction complex, resulting in reduced levels of nuclear β-catenin. At the functional level, JW74 induces reduced cell growth in all three tested cell lines, in part due to a delay in cell cycle progression and in part due to an induction of caspase-3-mediated apoptosis. Furthermore, JW74 induces differentiation in U2OS cells, which under standard conditions are resistant to osteogenic differentiation. JW74 also enhances differentiation of OS cell lines, which do not harbor a differentiation block. Interestingly, microRNAs (miRNAs) of the let-7 family, which are known tumor suppressors and inducers of differentiation, are significantly upregulated following treatment with JW74. We demonstrate for the first time that tankyrase inhibition triggers reduced cell growth and differentiation of OS cells. This may in part be due to an induction of let-7 miRNA. The presented data open for novel therapeutic strategies in the treatment of malignant OS.
    Cancer Medicine 02/2014; 3(1). DOI:10.1002/cam4.170 · 2.50 Impact Factor
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    • "Previously , Stegmeier lab revealed that Axin stability is regulated by Tankyrase and Tankyrase-mediated poly-ADP-ribose modification (PARsylation) of Axin is linked to Axin polyubiquitylation and subsequent degradation by the proteasome (Huang et al., 2009). Later, RNF146 was uncovered to be the E3 ligase for mediating Tankyrase-dependent degradation of Axin, thus playing a positive role in Wnt signaling (Callow et al., 2011; Zhang et al., 2011). Ubiquitin-specific protease 34 (USP34) is also reported to associate with Axin and control its levels, whereby modulating Wnt signaling (Lui et al., 2011). "
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    ABSTRACT: The Wnt signaling pathway plays crucial roles during embryonic development, whose aberration is implicated in a variety of human cancers. Axin, a key component of canonical Wnt pathway, plays dual roles in modulating Wnt signaling: on one hand, Axin scaffolds the "β-catenin destruction complex" to promote β-catenin degradation and therefore inhibits the Wnt signal transduction; on the other hand, Axin interacts with LRP5/6 and facilitates the recruitment of GSK3 to the plasma membrane to promote LRP5/6 phosphorylation and Wnt signaling. The differential assemblies of Axin with these two distinct complexes have to be tightly controlled for appropriate transduction of the "on" or "off" Wnt signal. So far, there are multiple mechanisms revealed in the regulation of Axin activity, such as post-transcriptional modulation, homo/hetero-polymerization and auto-inhibition. These mechanisms may work cooperatively to modulate the function of Axin, thereby playing an important role in controlling the canonical Wnt signaling. In this review, we will focus on the recent progresses regarding the regulation of Axin function in canonical Wnt signaling.
    Protein & Cell 01/2014; 5(3). DOI:10.1007/s13238-014-0019-2 · 3.25 Impact Factor
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