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Review
. 2017 Dec;174(24):4611-4636.
doi: 10.1111/bph.14038. Epub 2017 Nov 5.

Regulation of Wnt/β-catenin Signalling by Tankyrase-Dependent poly(ADP-ribosyl)ation and Scaffolding

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Free PMC article
Review

Regulation of Wnt/β-catenin Signalling by Tankyrase-Dependent poly(ADP-ribosyl)ation and Scaffolding

Laura Mariotti et al. Br J Pharmacol. .
Free PMC article

Abstract

The Wnt/β-catenin signalling pathway is pivotal for stem cell function and the control of cellular differentiation, both during embryonic development and tissue homeostasis in adults. Its activity is carefully controlled through the concerted interactions of concentration-limited pathway components and a wide range of post-translational modifications, including phosphorylation, ubiquitylation, sumoylation, poly(ADP-ribosyl)ation (PARylation) and acetylation. Regulation of Wnt/β-catenin signalling by PARylation was discovered relatively recently. The PARP tankyrase PARylates AXIN1/2, an essential central scaffolding protein in the β-catenin destruction complex, and targets it for degradation, thereby fine-tuning the responsiveness of cells to the Wnt signal. The past few years have not only seen much progress in our understanding of the molecular mechanisms by which PARylation controls the pathway but also witnessed the successful development of tankyrase inhibitors as tool compounds and promising agents for the therapy of Wnt-dependent dysfunctions, including colorectal cancer. Recent work has hinted at more complex roles of tankyrase in Wnt/β-catenin signalling as well as challenges and opportunities in the development of tankyrase inhibitors. Here we review some of the latest advances in our understanding of tankyrase function in the pathway and efforts to modulate tankyrase activity to re-tune Wnt/β-catenin signalling in colorectal cancer cells.

Linked articles: This article is part of a themed section on WNT Signalling: Mechanisms and Therapeutic Opportunities. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.24/issuetoc.

Figures

Figure 1
Figure 1
Roles of tankyrase‐dependent poly(ADP‐ribosyl)ation (PARylation) in Wnt/β‐catenin signalling. (A) Under basal Wnt/β‐catenin signalling conditions, PARylation by tankyrase limits the levels of AXIN. Following PARylation, AXIN is ubiquitylated by RNF146 and targeted for proteasomal degradation. (B) Upon Wnt stimulation, PARylated AXIN is stabilized. PARylation facilitates AXIN interaction with LRP5/6 in Wnt signalosomes. Note that AXIN, Dishevelled and tankyrase polymerize and APC dimerizes, and that this is a mechanistically important aspect of the dynamic signalling complexes (Fiedler et al., 2011; Kunttas‐Tatli et al., 2014; Mariotti et al., 2016). For simplicity, proteins are shown as monomers; higher‐order stoichiometry and multivalency are not reflected in the diagrams and nomenclature does not consider multiple paralogues of pathway components.
Figure 2
Figure 2
Scaffolding functions of tankyrase. (A) Domain organization of human TNKS and TNKS2. HPS, N‐terminal extension containing homopolymeric stretches of His, Pro and Ser; ARCs, ankyrin repeat clusters; SAM, sterile α motif domain; PARP, catalytic domain. The percentage identity of amino acids between TNKS and TNKS2 is specified for the indicated functional domains. (B) Domain organization of AXIN1. TBM, tankyrase‐binding motif; RGS, regulator of G‐protein signalling domain; DIX, polymerizing domain present in Dishevelled and AXIN (sometimes referred to as DAX domain in AXIN). Binding sites for other β‐catenin destruction complex components are indicated. (C) Multiple sequence alignment of AXIN orthologues/paralogues from the indicated species, coloured by percentage identity. The TBMs are indicated. Note that Drosophila Axin lacks the second TBM. The red asterisk denotes a V26D mutation identified in murine Axin2 (Qian et al., 2011). (D) Structural (surface and cartoon) representation of murine Tnks ARC2–3, bound to the murine Axin1 N‐terminus with two TBMs, shown in stick representation [protein data bank (PDB) code 3UTM] (Morrone et al., 2012). In the crystal, ARC2–3 forms a dimer in which both copies of ARC2 are bound by one of the two TBMs of Axin1, respectively. (E) Detailed structural representation of the Axin1 TBMs (with indicated amino acid positions) on Tnks ARC2. The figure was generated by superimposing both ARC2–3 copies onto each other and displaying ARC2 bound to TBM1. Despite the N‐terminal insertion in TBM2, the arginine (typically at position 1) occupies the same sub‐pocket on the ARC, resulting in a looping out of the intervening residues. (F) Structural (transparent surface and cartoon) representation of a TNKS SAM polymer observed by X‐ray crystallography (PDB code 5JU5) (Mariotti et al., 2016). (G) Avidity model for the interaction of AXIN and tankyrase, modified from Mariotti et al. (2016). Multivalency and polymerization of both tankyrase and AXIN enable avidity contributions in the interaction between both proteins. Note that tankyrase polymerization also promotes its PARP activity (Mariotti et al., 2016; Riccio et al., 2016).
Figure 3
Figure 3
β‐catenin degradasomes induced by TNKSi. SW480 CRC cells were treated with the TNKSi XAV939 and immunostained for AXIN2 (red) and transiently expressed epitope‐tagged wild‐type TNKS2 (TNKS2 WT) or a TNKS2 mutant variant deficient in substrate binding (through site‐directed mutation of ARCs 1, 2, 4 and 5) and polymerization (TNKS2 xx3xx VY903/920WA) (green). Yellow arrowheads indicate colocalization of TNKS2 and AXIN2 in β‐catenin degradasomes; red arrowheads indicate absence of colocalization for the scaffolding‐defective mutant variant of TNKS2. The figure was modified from Mariotti et al. (2016).
Figure 4
Figure 4
Allosteric regulation of RNF146/Iduna by PAR binding. (A) Structure of a linear PAR chain, here attached to Asp/Glu. The O‐glycosidic bonds linking ADP‐ribose units are highlighted. The green box indicates iso‐ADP‐ribose. Tankyrase is thought to generate linear PAR chains (Rippmann et al., 2002); PAR branches are therefore omitted. (B) Domain organization of human RNF146. Potential TBMs are indicated (DaRosa et al., 2015). The boxed area, which includes the isolated RING domain, corresponds to (C). (C) Structural representation of RNF146 bound to iso‐ADP‐ribose [protein data bank (PDB) code 4QPL] and the E2 enzyme UbcH5a (DaRosa et al., 2015). The isolated RING domain of RNF146 (PDB code 2D8T, one representative of the solution structure ensemble) is superimposed (DaRosa et al., 2015). Domains and corresponding Zn2+ ions are colour‐coded as in (B). Key residues involved in PAR coordination and the allosteric switch are shown in stick representation. Note the clash occurring between the RING domain (yellow) and the E2 enzyme (blue) in the absence of the PAR ligand, and the conformational change upon PAR binding, resulting in a reorientation of Trp65.
Figure 5
Figure 5
Binding modes of catalytic TNKSi and alternative inhibition strategy. (A) Structural representation of TNKS/TNKS2 PARP domain:inhibitor complexes. Residues of the catalytic H‐Y‐E triad are indicated. Adenosine and nicotinamide sites are highlighted with blue and orange dashed circles, respectively, in the left and central panels, respectively. Left, structure of the TNKS2 PARP domain with the adenosine site binder G007‐LK (shown in blue) [protein data bank (PDB) code 4HYF] (Voronkov et al., 2013). Loops lining the inhibitor binding site are indicated. Centre, structure of the TNKS PARP domain with the nicotinamide site binder XAV939 (shown in orange) (PDB code 3UH4) (Kirby et al., 2012). Right, structure of the TNKS PARP domain with a dual site binder (shown in green) (PDB code 4I9I) (Bregman et al., 2013b). The donor and acceptor sites are highlighted in magenta. (B) Structural representation of TNKS2 ARC4 (in surface representation) bound to two macrocyclized TBM peptides shown in superposition (PDB codes 5BXO and 5BXU) (Xu et al., 2017b). The peptide sequence is shown on the right with the position of the two different peptide staples, whose structures are shown. Amino acid positions of the TBM are indicated.
Figure 6
Figure 6
Potential determinants of TNKSi responses: APC mutation status and telomeric roles of tankyrase. (A) Schematic representation of APC with domains and motifs drawn to scale (see Stamos and Weis, 2013). So‐called 15‐ and 20‐amino‐acid‐repeats (15R and 20R) bind β‐catenin (except for 20R2), with the affinity of the 20Rs for β‐catenin being enhanced by their phosphorylation (Eklof Spink et al., 2001; Ha et al., 2004; Xing et al., 2004). SAMP repeats bind to AXIN1/2 (Spink et al., 2000). 20R2 and the catenin interaction domain (CID) / region B are required for β‐catenin ubiquitylation but do not bind β‐catenin; instead, they regulate AXIN1/2 binding to APC, and CID / region B is proposed to bind α‐catenin (Liu et al., 2006; Kohler et al., 2008; Choi et al., 2013; Pronobis et al., 2015). The mutation cluster region (MCR), a mutation hotspot in CRC (Kohler et al., 2008), is indicated in magenta. APC truncations observed in commonly used CRC cell lines are indicated by the arrows (Rowan et al., 2000; Ikediobi et al., 2006); labels are colour‐coded according to the indicated effects of TNKSi on AXIN and non‐phospho (active) β‐catenin levels and β‐catenin‐dependent transcription. *Note that the classification of DLD‐1 and HCT‐15 cells as TNKSi‐sensitive or ‐resistant varies between studies, given an ‘intermediate’ response (Huang et al., 2009; Lau et al., 2013; de la Roche et al., 2014; Tanaka et al., 2017). Very low AXIN1/2 levels in KM12 cells (Tanaka et al., 2017) may be responsible for non‐detectable AXIN accumulation upon tankyrase inhibition. (B) Multiple sequence alignment of the N‐termini of TERF1/TRF1 (telomeric repeat binding factor 1) orthologues from the indicated species, coloured by percentage identity. The amino acid numbering refers to human TERF1. The 8‐amino‐acid TBM is boxed in red. The murine Terf1 orthologue sequence is boxed in yellow and shows no conservation of the TBM.

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