In Wnt/β-catenin signaling, the β-catenin protein level is deliberately controlled by the assembly of the multiprotein β-catenin destruction complex composed of Axin, adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β), casein kinase 1α (CK1α), and others. Here we provide compelling evidence that formation of the destruction complex is driven by protein liquid-liquid phase separation (LLPS) of Axin. An intrinsically disordered region in Axin plays an important role in driving its LLPS. Phase-separated Axin provides a scaffold for recruiting GSK3β, CK1α, and β-catenin. APC also undergoes LLPS in vitro and enhances the size and dynamics of Axin phase droplets. The LLPS-driven assembly of the destruction complex facilitates β-catenin phosphorylation by GSK3β and is critical for the regulation of β-catenin protein stability and thus Wnt/β-catenin signaling.

Wnt signaling components have been shown to control key events in embryogenesis and to maintain tissue homeostasis in the adult. Nkd1/2 and Axin1/2 protein families are required for feedback regulation of Wnt signaling. The mechanisms by which Nkd1 and Nkd2 exhibit significant differences in signal transduction remain incompletely understood. Here we report that Rnf25/AO7, a previously identified E3 ubiquitin ligase for Nkd2, physically interacts with Nkd1 and Axin in an E3 ligase-independent manner to strengthen Wnt signalling. To determine the biological role of Rnf25 in vivo, we found that the renal mesenchymal cell, in which rnf25 was knocked-down, also exhibited more epithelial characters than MOCK control. Meanwhile, the transcriptional level of rnf25 was elevated in three separate tumor tissues more than that in paracarcinomatous tissue. Depletion of Rnf25 in zebrafish embryos attenuated transcriptions of maternal and zygotic Wnt target genes. Our results indicated that Rnf25 might serve as a molecular device, controlling the different antagonizing functions against canonical Wnt signaling between Nkd1 and Nkd2 cooperated with Axin.

Axin is a key scaffolding protein responsible for the formation of the β-catenin destruction complex. Stability of Axin protein is regulated by the ubiquitin-proteasome system, and modulation of cellular concentration of Axin protein has a profound effect on Wnt/β-catenin signaling. Although E3s promoting Axin ubiquitination have been identified, the deubiquitinase responsible for Axin deubiquitination and stabilization remains unknown. Here, we identify USP7 as a potent negative regulator of Wnt/β-catenin signaling through CRISPR screens. Genetic ablation or pharmacological inhibition of USP7 robustly increases Wnt/β-catenin signaling in multiple cellular systems. USP7 directly interacts with Axin through its TRAF domain, and promotes deubiquitination and stabilization of Axin. Inhibition of USP7 regulates osteoblast differentiation and adipocyte differentiation through increasing Wnt/β-catenin signaling. Our study reveals a critical mechanism that prevents excessive degradation of Axin and identifies USP7 as a target for sensitizing cells to Wnt/β-catenin signaling.

The Wnt/β-catenin pathway is critical to maintaining cell fate decisions. Recent study showed that liquid-liquid-phase separation (LLPS) of Axin organized the β-catenin destruction complex condensates in a normal cellular state. Mutations inactivating the APC gene are found in approximately 80% of all human colorectal cancer (CRC). However, the molecular mechanism of the formation of β-catenin destruction complex condensates organized by Axin phase separation and how APC mutations impact the condensates are still unclear. Here, we report that the β-catenin destruction complex, which is constructed by Axin, was assembled condensates via a phase separation process in CRC cells. The key role of wild-type APC is to stabilize destruction complex condensates. Surprisingly, truncated APC did not affect the formation of condensates, and GSK 3β and CK1α were unsuccessfully recruited, preventing β-catenin phosphorylation and resulting in accumulation in the cytoplasm of CRCs. Besides, we propose that the phase separation ability of Axin participates in the nucleus translocation of β-catenin and be incorporated and concentrated into transcriptional condensates, affecting the transcriptional activity of Wnt signaling pathway.

Deregulation of the Wnt signal transduction pathway underlies numerous congenital disorders and cancers. Axin, a concentration-limiting scaffold protein, facilitates assembly of a "destruction complex" that prevents signaling in the unstimulated state and a plasma membrane-associated "signalosome" that activates signaling following Wnt stimulation. In the classical model, Axin is cytoplasmic under basal conditions, but relocates to the cell membrane after Wnt exposure; however, due to the very low levels of endogenous Axin, this model is based largely on examination of Axin at supraphysiological levels. Here, we analyze the subcellular distribution of endogenous Drosophila Axin in vivo and find that a pool of Axin localizes to cell membrane proximal puncta even in the absence of Wnt stimulation. Axin localization in these puncta is dependent on the destruction complex component Adenomatous polyposis coli (Apc). In the unstimulated state, the membrane association of Axin increases its Tankyrase-dependent ADP-ribosylation and consequent proteasomal degradation to control its basal levels. Furthermore, Wnt stimulation does not result in a bulk redistribution of Axin from cytoplasmic to membrane pools, but causes an initial increase of Axin in both of these pools, with concomitant changes in two post-translational modifications, followed by Axin proteolysis hours later. Finally, the ADP-ribosylated Axin that increases rapidly following Wnt stimulation is membrane associated. We conclude that even in the unstimulated state, a pool of Axin forms membrane-proximal puncta that are dependent on Apc, and that membrane association regulates both Axin levels and Axin's role in the rapid activation of signaling that follows Wnt exposure.

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.

Insulin-stimulated glucose uptake by the glucose transporter GLUT4 plays a central role in whole-body glucose homeostasis, dysregulation of which leads to type 2 diabetes. However, the molecular components and mechanisms regulating insulin-stimulated glucose uptake remain largely unclear. Here, we demonstrate that Axin interacts with the ADP-ribosylase tankyrase 2 (TNKS2) and the kinesin motor protein KIF3A, forming a ternary complex crucial for GLUT4 translocation in response to insulin. Specific knockdown of the individual components of the complex attenuated insulin-stimulated GLUT4 translocation to the plasma membrane. Importantly, TNKS2(-/-) mice exhibit reduced insulin sensitivity and higher blood glucose levels when re-fed after fasting. Mechanistically, we demonstrate that in the absence of insulin, Axin, TNKS and KIF3A are co-localized with GLUT4 on the trans-Golgi network. Insulin treatment suppresses the ADP-ribosylase activity of TNKS, leading to a reduction in ADP ribosylation and ubiquitination of both Axin and TNKS, and a concurrent stabilization of the complex. Inhibition of Akt, the major effector kinase of insulin signaling, abrogates the insulin-mediated complex stabilization. We have thus elucidated a new protein complex that is directly associated with the motor protein kinesin in insulin-stimulated GLUT4 translocation.

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.

Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ss-catenin pathway.

The Wnt/β-catenin signaling pathway plays essential roles in embryonic development and adult tissue homeostasis. Axin is a concentration-limiting factor responsible for the formation of the β-catenin destruction complex. Wnt signaling itself promotes the degradation of Axin. However, the underlying molecular mechanism and biological relevance of this targeting of Axin have not been elucidated. Here, we identify SIAH1/2 (SIAH) as the E3 ligase mediating Wnt-induced Axin degradation. SIAH proteins promote the ubiquitination and proteasomal degradation of Axin through interacting with a VxP motif in the GSK3-binding domain of Axin, and this function of SIAH is counteracted by GSK3 binding to Axin. Structural analysis reveals that the Axin segment responsible for SIAH binding is also involved in GSK3 binding but adopts distinct conformations in Axin/SIAH and Axin/GSK3 complexes. Knockout of SIAH1 blocks Wnt-induced Axin ubiquitination and attenuates Wnt-induced β-catenin stabilization. Our data suggest that Wnt-induced dissociation of the Axin/GSK3 complex allows SIAH to interact with Axin not associated with GSK3 and promote its degradation and that SIAH-mediated Axin degradation represents an important feed-forward mechanism to achieve sustained Wnt/β-catenin signaling.

Axin is a critical component of the β-catenin destruction complex and is also necessary for Wnt signaling initiation at the level of co-receptor activation. Axin contains an RGS domain, which is similar to that of proteins that accelerate the GTPase activity of heterotrimeric Gα/Gna proteins and thereby limit the duration of active G-protein signaling. Although G-proteins are increasingly recognized as essential components of Wnt signaling, it has been unclear whether this domain of Axin might function in G-protein regulation. This study was performed to test the hypothesis that Axin RGS-Gna interactions would be required to attenuate Wnt signaling. We tested these ideas using an axin1 genetic mutant (masterblind) and antisense oligo knockdowns in developing zebrafish and Xenopus embryos. We generated a point mutation that is predicted to reduce Axin-Gna interaction and tested for the ability of the mutant forms to rescue Axin loss-of-function function. This Axin point mutation was deficient in binding to Gna proteins in vitro, and was unable to relocalize to the plasma membrane upon Gna overexpression. We found that the Axin point mutant construct failed to rescue normal anteroposterior neural patterning in masterblind mutant zebrafish, suggesting a requirement for G-protein interactions in this context. We also found that the same mutant was able to rescue deficiencies in maternal axin1 loss-of-function in Xenopus. These data suggest that maternal and zygotic Wnt signaling may differ in the extent of Axin regulation of G-protein signaling. We further report that expression of a membrane-localized Axin construct is sufficient to inhibit Wnt/β-catenin signaling and to promote Axin protein turnover.

The Wnt/β-catenin pathway is a highly conserved, frequently mutated developmental and cancer pathway. Its output is defined mainly by β-catenin's phosphorylation- and ubiquitylation-dependent proteasomal degradation, initiated by the multi-protein β-catenin destruction complex. The precise mechanisms underlying destruction complex function have remained unknown, largely because of the lack of suitable in vitro systems. Here we describe the in vitro reconstitution of an active human β-catenin destruction complex from purified components, recapitulating complex assembly, β-catenin modification, and degradation. We reveal that AXIN1 polymerization and APC promote β-catenin capture, phosphorylation, and ubiquitylation. APC facilitates β-catenin's flux through the complex by limiting ubiquitylation processivity and directly interacts with the SCFβ-TrCP E3 ligase complex in a β-TrCP-dependent manner. Oncogenic APC truncation variants, although part of the complex, are functionally impaired. Nonetheless, even the most severely truncated APC variant promotes β-catenin recruitment. These findings exemplify the power of biochemical reconstitution to interrogate the molecular mechanisms of Wnt/β-catenin signaling.

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.

Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis and is altered in human cancer. Signaling is turned on and off by regulating stability of the effector β-catenin (β-cat). The multiprotein destruction complex binds and phosphorylates β-cat and transfers it to the SCF-TrCP E3-ubiquitin ligase for ubiquitination and destruction. Wnt signals act though Dishevelled to turn down the destruction complex, stabilizing β-cat. Recent work clarified underlying mechanisms, but important questions remain. We explore β-cat transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin. We find that Slimb/TrCP is a dynamic component of the destruction complex biomolecular condensate, while other E3 proteins are not. Recruitment requires Axin and not APC, and Axin's RGS domain plays an important role. We find that elevating Dishevelled levels in Drosophila embryos has paradoxical effects, promoting the ability of limiting levels of Axin to turn off Wnt signaling. When we elevate Dishevelled levels, it forms its own cytoplasmic puncta, but these do not recruit Axin. Superresolution imaging in mammalian cells raises the possibility that this may result by promoting Dishevelled:Dishevelled interactions at the expense of Dishevelled: Axin interactions when Dishevelled levels are high.

Axin and the adenomatous polyposis coli (APC) tumor suppressor protein are components of the Wnt/Wingless growth factor signaling pathway. In the absence of Wnt signal, Axin and APC regulate cytoplasmic levels of the proto-oncogene beta-catenin through the formation of a large complex containing these three proteins, glycogen synthase kinase 3beta (GSK3beta) and several other proteins. Both Axin and APC are known to be critical for beta-catenin regulation, and truncations in APC that eliminate the Axin-binding site result in human cancers. A protease-resistant domain of Axin that contains the APC-binding site is a member of the regulators of G-protein signaling (RGS) superfamily. The crystal structures of this domain alone and in complex with an Axin-binding sequence from APC reveal that the Axin-APC interaction occurs at a conserved groove on a face of the protein that is distinct from the G-protein interface of classical RGS proteins. The molecular interactions observed in the Axin-APC complex provide a rationale for the evolutionary conservation seen in both proteins.

Dishevelled exerts a molecular force that guides cell fate, but how it does so remains enigmatic. In this issue, Kang et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202205069) show Dvl2 undergoes liquid-liquid phase separation to stabilize β-catenin by pulling Axin into its biomolecular condensate at the plasma membrane.

Wnts and the components of Wnt/β-catenin signaling are widely expressed in midbrain and required to control the fate specification of dopaminergic (DAergic) neurons, a neuronal population that specifically degenerate in Parkinson's disease (PD). Accumulating evidence suggest that mitochondrial dysfunction plays a key role in pathogenesis of PD. Axin-2, a negative regulator of Wnt/β-catenin signaling affects mitochondrial biogenesis and death/birth of new DAergic neurons is not fully explored. We investigated the functional role of Axin-2/Wnt/β-catenin signaling in mitochondrial biogenesis and DAergic neurogenesis in 6-hydroxydopamine (6-OHDA) induced rat model of PD-like phenotypes. We demonstrate that single unilateral injection of 6-OHDA into the medial forebrain bundle (MFB) potentially dysregulates Wnt/β-catenin signaling in substantia nigra pars compacta (SNpc). We used shRNA lentiviruses to genetically knockdown Axin-2 to up-regulate Wnt/β-catenin signaling in SNpc in parkinsonian rats. Genetic knockdown of Axin-2 up-regulates Wnt/β-catenin signaling by destabilizing the β-catenin degradation complex in SNpc in parkinsonian rats. Axin-2 shRNA mediated activation of Wnt/β-catenin signaling improved behavioural functions and protected the nigral DAergic neurons by increasing mitochondrial functionality in parkinsonian rats. Axin-2 shRNA treatment reduced apoptotic signaling, autophagy and ROS generation and improved mitochondrial membrane potential which promotes mitochondrial biogenesis in SNpc in parkinsonian rats. Interestingly, Axin-2 shRNA-mediated up-regulation of Wnt/β-catenin signaling enhanced net DAergic neurogenesis by regulating proneural genes (Nurr-1, Pitx-3, Ngn-2, and NeuroD1) and mitochondrial biogenesis in SNpc in parkinsonian rats. Therefore, our data suggest that pharmacological/genetic manipulation of Wnt signaling that enhances the endogenous regenerative capacity of DAergic neurons may have implication for regenerative approaches in PD.

Axin (also known as AXIN1) is a central negative regulator of the proto-oncogenic Wnt/β-catenin signaling pathway, as axin condensates provide a scaffold for the assembly of a multiprotein complex degrading β-catenin. Axin, in turn, is degraded through tankyrase. Consequently, tankyrase small-molecule inhibitors block Wnt signaling by stabilizing axin, revealing potential for cancer therapy. Here, we discovered that axin is phosphorylated by casein kinase 1 alpha 1 (CSNK1A1, also known as CK1α) at an N-terminal casein kinase 1 consensus motif, and that this phosphorylation is antagonized by the catalytic subunit alpha of protein phosphatase 1 (PPP1CA, hereafter referred to as PP1). Axin condensates promoted phosphorylation by enriching CK1α over PP1. Importantly, the phosphorylation took place within the tankyrase-binding site, electrostatically and/or sterically hindering axin-tankyrase interaction, and counteracting tankyrase-mediated degradation of axin. Thus, the presented data propose a novel mechanism regulating axin stability, with implications for Wnt signaling, cancer therapy and self-organization of biomolecular condensates.

Multiple signaling pathways regulate the kinase GSK3β by inhibitory phosphorylation at Ser9, which then occupies the GSK3β priming pocket and blocks substrate binding. Since this mechanism should affect GSK3β activity toward all primed substrates, it is unclear why Ser9 phosphorylation does not affect other GSK3β-dependent pathways, such as Wnt signaling. We used biochemical reconstitution and cell culture assays to evaluate how Wnt-associated GSK3β is insulated from cross-activation by other signals. We found that the Wnt-specific scaffold protein Axin allosterically protects GSK3β from phosphorylation at Ser9 by upstream kinases, which prevents accumulation of pS9-GSK3β in the Axin•GSK3β complex. Scaffold proteins that protect bound proteins from alternative pathway reactions could provide a general mechanism to insulate signaling pathways from improper crosstalk.

In Wnt/β-catenin signaling, the transcriptional coactivator β-catenin is regulated by its phosphorylation in a complex that includes the scaffold protein Axin and associated kinases. Wnt binding to its coreceptors activates the cytosolic effector Dishevelled (Dvl), leading to the recruitment of Axin and the inhibition of β-catenin phosphorylation. This process requires interaction of homologous DIX domains present in Dvl and Axin, but is mechanistically undefined. We show that Dvl DIX forms antiparallel, double-stranded oligomers in vitro, and that Dvl in cells forms oligomers typically <10 molecules at endogenous expression levels. Axin DIX (DAX) forms small single-stranded oligomers, but its self-association is stronger than that of DIX. DAX caps the ends of DIX oligomers, such that a DIX oligomer has at most four DAX binding sites. The relative affinities and stoichiometry of the DIX-DAX interaction provide a mechanism for efficient inhibition of β-catenin phosphorylation upon Axin recruitment to the Wnt receptor complex.