AXIN2 Reduces the Survival of Porcine Induced Pluripotent Stem Cells (piPSCs).

The establishment of porcine pluripotent stem cells (piPSCs) is critical but remains challenging. All piPSCs are extremely sensitive to minor perturbations of culture conditions and signaling network. Inhibitors, such as CHIR99021 and XAV939 targeting the WNT signaling pathway, have been added in a culture medium to modify the cell regulatory network. However, potential side effects of inhibitors could confine the pluripotency and practicability of piPSCs. This study aimed to investigate the roles of AXIN, one component of the WNT pathway in piPSCs. Here, porcine AXIN1 and AXIN2 genes were knocked-down or overexpressed. Digital RNA-seq was performed to explore the mechanism of cell proliferation and apoptosis. We found that (1) overexpression of the porcine AXIN2 gene significantly reduced survival and negatively impacted the pluripotency of piPSCs, and (2) knockdown of AXIN2, a negative effector of the WNT signaling pathway, enhanced the expression of genes involved in cell cycle but reduced the expression of genes related to cell differentiation, death, and apoptosis.

An AMPK/Axin1-Rac1 signaling pathway mediates contraction-regulated glucose uptake in skeletal muscle cells.

Contraction stimulates skeletal muscle glucose uptake predominantly through activation of AMP-activated protein kinase (AMPK) and Rac1. However, the molecular details of how contraction activates these signaling proteins are not clear. Recently, Axin1 has been shown to form a complex with AMPK and liver kinase B1 during glucose starvation-dependent activation of AMPK. Here, we demonstrate that electrical pulse-stimulated (EPS) contraction of C2C12 myotubes or treadmill exercise of C57BL/6 mice enhanced reciprocal coimmunoprecipitation of Axin1 and AMPK from myotube lysates or gastrocnemius muscle tissue. Interestingly, EPS or exercise upregulated total cellular Axin1 levels in an AMPK-dependent manner in C2C12 myotubes and gastrocnemius mouse muscle, respectively. Also, direct activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide treatment of C2C12 myotubes or gastrocnemius muscle elevated Axin1 protein levels. On the other hand, siRNA-mediated Axin1 knockdown lessened activation of AMPK in contracted myotubes. Further, AMPK inhibition with compound C or siRNA-mediated knockdown of AMPK or Axin1 blocked contraction-induced GTP loading of Rac1, p21-activated kinase phosphorylation, and contraction-stimulated glucose uptake. In summary, our results suggest that an AMPK/Axin1-Rac1 signaling pathway mediates contraction-stimulated skeletal muscle glucose uptake.

PTHR1 May Be Involved in Progression of Osteosarcoma by Regulating miR-124-3p-AR-Tgfb1i1, miR-27a-3p-PPARG-Abca1, and miR-103/590-3p-AXIN2 Axes.

Our previous study has indicated that the parathyroid hormone type 1 receptor (PTHR1) may play important roles in development and progression of osteosarcoma (OS) by regulating Wnt, angiogenesis, and inflammation pathway genes. The goal of this study was to further illuminate the roles of PTHR1 in OS by investigating upstream regulation mechanisms (including microRNA [miRNA] and transcription factors [TFs]) of crucial genes. The microarray dataset GSE46861 was downloaded from the Gene Expression Omnibus database, in which six tumors with short hairpin RNA (shRNA) PTHR1 knockdown (PTHR1.358) and six tumors with shRNA control knockdown (Ren.1309) were collected from mice. Differentially expressed genes (DEGs) between PTHR1.358 and Ren.1309 were identified using the linear models for microarray data (LIMMA) method, and then the miRNA-TF-mRNA regulatory network was constructed using data from corresponding databases, followed by module analysis, to screen crucial regulatory relationships. OS-related human miRNAs were extracted from the curated Osteosarcoma Database. Gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool. As a result, the miRNA-TF-mRNA regulatory network, including 1049 nodes (516 miRNA, 25 TFs, and 508 DEGs) and 15942 edges (interaction relationships, such as Pparg-Abca1 and miR-590-3p-AXIN2), was constructed, from which three significant modules were extracted and modules 2 and 3 contained interactions between miRNAs/TFs and DEGs such as miR-103-3p-AXIN2, miR-124-3p-AR-Tgfb1i1, and miR-27a-3p-PPARG-Abca1. miR-27a-3p was a known miRNA associated with OS. Abca1, AR, and miR-124-3p were hub genes in the miRNA-TF-mRNA network. Tgfb1i1 was involved in cell proliferation, Abca1 participated in the cholesterol metabolic process, and AXIN2 was associated with the canonical Wnt signaling pathway. Furthermore, we also confirmed upregulation of miR-590-3p and downregulation of AXIN2 in the mouse OS cell line K7M2-WT transfected with PTHR1 shRNA. In conclusion, PTHR1 may play important roles in progression of OS by activating miR-124-3p-AR-Tgfb1i1, miR-27a-3p-PPARG-Abca1, and miR-103/590-3p-AXIN2 axes.

Disruption of the Extracellular Matrix Progressively Impairs Central Nervous System Vascular Maturation Downstream of ß-Catenin Signaling.

Objective- The Wnt/ß-catenin pathway orchestrates development of the blood-brain barrier, but the downstream mechanisms involved at different developmental windows and in different central nervous system (CNS) tissues have remained elusive. Approach and Results- Here, we create a new mouse model allowing spatiotemporal investigations of Wnt/ß-catenin signaling by induced overexpression of Axin1, an inhibitor of ß-catenin signaling, specifically in endothelial cells ( Axin1 iEC- OE). AOE (Axin1 overexpression) in Axin1 iEC- OE mice at stages following the initial vascular invasion of the CNS did not impair angiogenesis but led to premature vascular regression followed by progressive dilation and inhibition of vascular maturation resulting in forebrain-specific hemorrhage 4 days post-AOE. Analysis of the temporal Wnt/ß-catenin driven CNS vascular development in zebrafish also suggested that Axin1 iEC- OE led to CNS vascular regression and impaired maturation but not inhibition of ongoing angiogenesis within the CNS. Transcriptomic profiling of isolated, ß-catenin signaling-deficient endothelial cells during early blood-brain barrier-development (E11.5) revealed ECM (extracellular matrix) proteins as one of the most severely deregulated clusters. Among the 20 genes constituting the forebrain endothelial cell-specific response signature, 8 ( Adamtsl2, Apod, Ctsw, Htra3, Pglyrp1, Spock2, Ttyh2, and Wfdc1) encoded bona fide ECM proteins. This specific ß-catenin-responsive ECM signature was also repressed in Axin1 iEC- OE and endothelial cell-specific ß-catenin-knockout mice ( Ctnnb1-KOiEC) during initial blood-brain barrier maturation (E14.5), consistent with an important role of Wnt/ß-catenin signaling in orchestrating the development of the forebrain vascular ECM. Conclusions- These results suggest a novel mechanism of establishing a CNS endothelium-specific ECM signature downstream of Wnt-ß-catenin that impact spatiotemporally on blood-brain barrier differentiation during forebrain vessel development. Visual Overview- An online visual overview is available for this article.

The Association Between AXIN2 Gene Polymorphisms and the Risk of Breast Cancer in Chinese Women.

Background: The protein AXIN2 is involved in the negative feedback regulation of the Wnt/ß-catenin signaling pathway; it functions by promoting ß-catenin degradation. AXIN2 mutations have been studied in various cancers. In this study, we genotyped three single nucleotide polymorphisms in the AXIN2 gene and investigated their association with the risk of breast cancer (BC) in the Chinese Han population. Methods: In a population of 415 BC patients and 528 controls the expression of AXIN2 was measured using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and compared with the overall survival (OS) of BC patients analyzed through Oncomine and Kaplan-Meier plotter databases. Bioinformatic analyses demonstrated that AXIN2 mRNA levels were downregulated in BC patients; this in turn correlated with a poorer survival rate for BC patients. Results: The polymorphisms rs11079571 and rs3923087, but not rs3923086, were associated with an increased risk of BC. The minor allele containing genotypes of polymorphism rs3923087 were positively associated with lymph node metastases. A haplotype analysis demonstrated that the ATA haplotype was correlated with an increased risk of BC. Conclusion: In conclusion, the downregulation of AXIN2 is related to poorer OS for BC patients. Its polymorphisms rs11079571 and rs3923087 confer susceptibility to BC. These findings should be confirmed with larger studies that include more diverse ethnic populations.

Axis inhibition protein 1 (Axin1) Deletion-Induced Hepatocarcinogenesis Requires Intact ß-Catenin but Not Notch Cascade in Mice.

Inactivating mutations of axis inhibition protein 1 (AXIN1), a negative regulator of the Wnt/ß-Catenin cascade, are among the common genetic events in human hepatocellular carcinoma (HCC), affecting approximately 10% of cases. In the present manuscript, we sought to define the genetic crosstalk between Axin1 mutants and Wnt/ß-catenin as well as Notch signaling cascades along hepatocarcinogenesis. We discovered that c-MET activation and AXIN1 mutations occur concomitantly in ~3%-5% of human HCC samples. Subsequently, we generated a murine HCC model by means of CRISPR/Cas9-based gene deletion of Axin1 (sgAxin1) in combination with transposon-based expression of c-Met in the mouse liver (c-Met/sgAxin1). Global gene expression analysis of mouse normal liver, HCCs induced by c-Met/sgAxin1, and HCCs induced by c-Met/∆N90-ß-Catenin revealed activation of the Wnt/ß-Catenin and Notch signaling in c-Met/sgAxin1 HCCs. However, only a few of the canonical Wnt/ß-Catenin target genes were induced in c-Met/sgAxin1 HCC when compared with corresponding lesions from c-Met/∆N90-ß-Catenin mice. To study whether endogenous ß-Catenin is required for c-Met/sgAxin1-driven HCC development, we expressed c-Met/sgAxin1 in liver-specific Ctnnb1 null mice, which completely prevented HCC development. Consistently, in AXIN1 mutant or null human HCC cell lines, silencing of ß-Catenin strongly inhibited cell proliferation. In striking contrast, blocking the Notch cascade through expression of either the dominant negative form of the recombinant signal-binding protein for immunoglobulin kappa J region (RBP-J) or the ablation of Notch2 did not significantly affect c-Met/sgAxin1-driven hepatocarcinogenesis. Conclusion: We demonstrated here that loss of Axin1 cooperates with c-Met to induce HCC in mice, in a ß-Catenin signaling-dependent but Notch cascade-independent way.

Heterogeneous nuclear ribonucleoprotein M promotes the progression of breast cancer by regulating the axin/ß-catenin signaling pathway.

Despite significant progress in the treatment of breast cancer due to advances in surgery, cytotoxic agents, and endocrine therapy, the prognosis for patients has not improved much. Accumulated evidence indicates that heterogeneous nuclear ribonucleoprotein M (hnRNPM) and Wnt/ß-catenin function as tumor oncogenes in the progression of many cancers. The present study aimed to explore whether HnRNPM/ß-catenin signaling molecules might serve as a genetic target for breast cancer treatment. To shed light on this issue, quantitative real-time polymerase chain reaction (qRT-PCR) detection, Western blotting, and immunohistochemical staining were performed. The hnRNPM is expressed at a much higher level in breast cancer tissues and cell lines than in noncancerous tissues and cell lines. In vitro studies revealed that overexpressed hnRNPM promoted cell proliferation and colony formation but inhibited cell apoptosis. In vivo results demonstrated that upregulation of hnRNPM dramatically increased breast cancer xenograft tumor growth. Western blotting and immunofluorescence studies revealed that hnRNPM markedly activated the Wnt/ß-catenin pathway and catalyzed its translocation from the cytoplasm to the nucleus by targeting axin, a negative regulator of Wnt/ß-catenin signaling in MCF-7 and KPL-4 cells. Elevated levels of c-Myc and cyclin D1 were observed when MCF-7 and KPL-4 cells were transfected with a hnRNPM vector. These findings indicate that the hnRNPM/axin/ß-catenin signaling pathway acts as an oncogenic promoter in the progression of breast cancer, suggesting that hnRNPM may be a potential target for the treatment of this disease.

Transforming Growth Factor ß Activation Primes Canonical Wnt Signaling Through Down-Regulation of Axin-2.

OBJECTIVE: Aberrant activation of Wnt signaling has been observed in tissues from patients with systemic sclerosis (SSc). This study aimed to determine the role of transforming growth factor ß (TGFß) in driving the increased Wnt signaling, through modulation of axis inhibition protein 2 (Axin-2), a critical regulator of the Wnt canonical pathway. METHODS: Canonical Wnt signaling activation was analyzed by TOPflash T cell factor/lymphoid enhancer factor promoter assays. Axin-2 was evaluated in vitro by analysis of Axin-2 primary/mature transcript expression and decay, TGFß receptor type I (TGFßRI) blockade, small interfering RNA-mediated depletion of tristetraprolin 1, and XAV-939-mediated Axin-2 stabilization. In vivo, Axin-2 messenger RNA (mRNA) and protein expression was determined in skin and lung biopsy samples from mice that express a kinase-deficient TGFßRII specifically on fibroblasts (TßRIIΔk-fib-transgenic mice) and from littermate controls. RESULTS: SSc fibroblasts displayed an increased response to canonical Wnt ligands despite basal levels of Wnt signaling that were comparable to those in healthy control fibroblasts in vitro. Notably, we showed that SSc fibroblasts had reduced basal expression of Axin-2, which was caused by an endogenous TGFß-dependent increase in Axin-2 mRNA decay. Accordingly, we observed that TGFß decreased Axin-2 expression both in vitro in healthy control fibroblasts and in vivo in TßRIIΔk-fib-transgenic mice. Additionally, using Axin-2 gain- and loss-of-function experiments, we demonstrated that the TGFß-induced increased response to Wnt activation characteristic of SSc fibroblasts depended on reduced bioavailability of Axin-2. CONCLUSION: This study highlights the importance of reduced bioavailability of Axin-2 in mediating the increased canonical Wnt response observed in SSc fibroblasts. This novel mechanism extends our understanding of the processes involved in Wnt/ß-catenin-driven pathology and supports the rationale for targeting the TGFß pathway to regulate the aberrant Wnt signaling observed during fibrosis.

Differential Roles of AXIN1 and AXIN2 in Tankyrase Inhibitor-Induced Formation of Degradasomes and ß-Catenin Degradation.

Inhibition of the tankyrase enzymes (TNKS1 and TNKS2) has recently been shown to induce highly dynamic assemblies of ß-catenin destruction complex components known as degradasomes, which promote degradation of ß-catenin and reduced Wnt signaling activity in colorectal cancer cells. AXIN1 and AXIN2/Conductin, the rate-limiting factors for the stability and function of endogenous destruction complexes, are stabilized upon TNKS inhibition due to abrogated degradation of AXIN by the proteasome. Since the role of AXIN1 versus AXIN2 as scaffolding proteins in the Wnt signaling pathway still remains incompletely understood, we sought to elucidate their relative contribution in the formation of degradasomes, as these protein assemblies most likely represent the morphological and functional correlates of endogenous ß-catenin destruction complexes. In SW480 colorectal cancer cells treated with the tankyrase inhibitor (TNKSi) G007-LK we found that AXIN1 was not required for degradasome formation. In contrast, the formation of degradasomes as well as their capacity to degrade ß-catenin were considerably impaired in G007-LK-treated cells depleted of AXIN2. These findings give novel insights into differential functional roles of AXIN1 versus AXIN2 in the ß-catenin destruction complex.

Reconstituting regulation of the canonical Wnt pathway by engineering a minimal ß-catenin destruction machine.

Negatively regulating key signaling pathways is critical to development and altered in cancer. Wnt signaling is kept off by the destruction complex, which is assembled around the tumor suppressors APC and Axin and targets ß-catenin for destruction. Axin and APC are large proteins with many domains and motifs that bind other partners. We hypothesized that if we identified the essential regions required for APC:Axin cooperative function and used these data to design a minimal ß-catenin-destruction machine, we would gain new insights into the core mechanisms of destruction complex function. We identified five key domains/motifs in APC or Axin that are essential for their function in reconstituting Wnt regulation. Strikingly, however, certain APC and Axin mutants that are nonfunctional on their own can complement one another in reducing ß-catenin, revealing that the APC:Axin complex is a highly robust machine. We used these insights to design a minimal ß-catenin-destruction machine, revealing that a minimized chimeric protein covalently linking the five essential regions of APC and Axin reconstitutes destruction complex internal structure, size, and dynamics, restoring efficient ß-catenin destruction in colorectal tumor cells. On the basis of our data, we propose a new model of the mechanistic function of the destruction complex as an integrated machine.

Axin Regulates Dendritic Spine Morphogenesis through Cdc42-Dependent Signaling.

During development, scaffold proteins serve as important platforms for orchestrating signaling complexes to transduce extracellular stimuli into intracellular responses that regulate dendritic spine morphology and function. Axin ("axis inhibitor") is a key scaffold protein in canonical Wnt signaling that interacts with specific synaptic proteins. However, the cellular functions of these protein-protein interactions in dendritic spine morphology and synaptic regulation are unclear. Here, we report that Axin protein is enriched in synaptic fractions, colocalizes with the postsynaptic marker PSD-95 in cultured hippocampal neurons, and interacts with a signaling protein Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in synaptosomal fractions. Axin depletion by shRNA in cultured neurons or intact hippocampal CA1 regions significantly reduced dendritic spine density. Intriguingly, the defective dendritic spine morphogenesis in Axin-knockdown neurons could be restored by overexpression of the small Rho-GTPase Cdc42, whose activity is regulated by CaMKII. Moreover, pharmacological stabilization of Axin resulted in increased dendritic spine number and spontaneous neurotransmission, while Axin stabilization in hippocampal neurons reduced the elimination of dendritic spines. Taken together, our findings suggest that Axin promotes dendritic spine stabilization through Cdc42-dependent cytoskeletal reorganization.

Different requirements for Wnt signaling in tongue myogenic subpopulations.

The tongue is a muscular organ that is essential in vertebrates for important functions, such as food intake and communication. Little is known about regulation of myogenic progenitors during tongue development when compared with the limb or trunk region. In this study, we investigated the relationship between different myogenic subpopulations and the function of canonical Wnt signaling in regulating these subpopulations. We found that Myf5- and MyoD-expressing myogenic subpopulations exist during embryonic tongue myogenesis. In the Myf5-expressing myogenic progenitors, there is a cell-autonomous requirement for canonical Wnt signaling for cell migration and differentiation. In contrast, the MyoD-expressing subpopulation does not require canonical Wnt signaling during tongue myogenesis. Taken together, our results demonstrate that canonical Wnt signaling differentially regulates the Myf5- and MyoD-expressing subpopulations during tongue myogenesis.

The roles of AXIN2 in tumorigenesis and epigenetic regulation.

AXIN2, an important regulator in Wnt/ß-catenin signaling pathway, takes part in regulating cell proliferation, cytometaplasia, migration, apoptosis and other important functions, has showed close relations with the development of liver cancer, colon cancer, lung cancer, breast cancer and so on. The epigenetic regulation provides new insights for further exploring the pathogenesis of tumor. In this paper, the roles of AXIN2 in tumorigenesis, AXIN2 methylation, ubiquitination and siRNA/RNA regulation will be reviewed.

Improving oral implant osseointegration in a murine model via Wnt signal amplification.

AIM: To determine the key biological events occurring during implant failure and then we use this knowledge to develop new biology-based strategies that improve osseointegration. MATERIALS AND METHODS: Wild-type and Axin2(LacZ/LacZ) adult male mice underwent oral implant placement, with and without primary stability. Peri-implant tissues were evaluated using histology, alkaline phosphatase (ALP) activity, tartrate resistant acid phosphatase (TRAP) activity and TUNEL staining. In addition, mineralization sites, collagenous matrix organization and the expression of bone markers in the peri-implant tissues were assessed. RESULTS: Maxillary implants lacking primary stability show histological evidence of persistent fibrous encapsulation and mobility, which recapitulates the clinical problems of implant failure. Despite histological and molecular evidence of fibrous encapsulation, osteoblasts in the gap interface exhibit robust ALP activity. This mineralization activity is counteracted by osteoclast activity that resorbs any new bony matrix and consequently, the fibrous encapsulation remains. Using a genetic mouse model, we show that implants lacking primary stability undergo osseointegration, provided that Wnt signalling is amplified. CONCLUSIONS: In a mouse model of oral implant failure caused by a lack of primary stability, we find evidence of active mineralization. This mineralization, however, is outpaced by robust bone resorption, which culminates in persistent fibrous encapsulation of the implant. Fibrous encapsulation can be prevented and osseointegration assured if Wnt signalling is elevated at the time of implant placement.

Axin: an emerging key scaffold at the synapse.

Neurons communicate through neurotransmission at the synapse. Precise regulation of the synaptic structure and signaling during the formation and remodeling of synapses is vital for information processing between neurons. Scaffold proteins play key roles in synapses by tethering the signaling cascades spatially and temporally to ensure proper brain functioning. This review summarizes the recent evidence indicating that Axin, a scaffold protein, plays a central role in orchestrating presynaptic and postsynaptic signaling complexes to regulate synapse development and plasticity in the central nervous system.

Long noncoding RNAs associated with liver regeneration 1 accelerates hepatocyte proliferation during liver regeneration by activating Wnt/ß-catenin signaling.

UNLABELLED: In recent years, long noncoding RNAs (lncRNAs) have been investigated as a new class of regulators of biological function. A recent study reported that lncRNAs control cell proliferation in hepatocellular carcinoma (HCC). However, the role of lncRNAs in liver regeneration and the overall mechanisms remain largely unknown. To address this issue, we carried out a genome-wide lncRNA microarray analysis during liver regeneration in mice after 2/3 partial hepatectomy (PH) at various timepoints. The results revealed differential expression of a subset of lncRNAs, notably a specific differentially expressed lncRNA associated with Wnt/ß-catenin signaling during liver regeneration (an lncRNA associated with liver regeneration, termed lncRNA-LALR1). The functions of lncRNA-LALR1 were assessed by silencing and overexpressing this lncRNA in vitro and in vivo. We found that lncRNA-LALR1 enhanced hepatocyte proliferation by promoting progression of the cell cycle in vitro. Furthermore, we showed that lncRNA-LALR1 accelerated mouse hepatocyte proliferation and cell cycle progression during liver regeneration in vivo. Mechanistically, we discovered that lncRNA-LALR1 facilitated cyclin D1 expression through activation of Wnt/ß-catenin signaling by way of suppression of Axin1. In addition, lncRNA-LALR1 inhibited the expression of Axin1 mainly by recruiting CTCF to the AXIN1 promoter region. We also identified a human ortholog RNA of lncRNA-LALR1 (lncRNA-hLALR1) and found that it was expressed in human liver tissues. CONCLUSION: lncRNA-LALR1 promotes cell cycle progression and accelerates hepatocyte proliferation during liver regeneration by activating Wnt/ß-catenin signaling. Pharmacological intervention targeting lncRNA-LALR1 may be therapeutically beneficial in liver failure and liver transplantation by inducing liver regeneration.

Runx2 protein represses Axin2 expression in osteoblasts and is required for craniosynostosis in Axin2-deficient mice.

Runx2 and Axin2 regulate craniofacial development and skeletal maintenance. Runx2 is essential for calvarial bone development, as Runx2 haploinsufficiency causes cleidocranial dysplasia. In contrast, Axin2-deficient mice develop craniosynostosis because of high ß-catenin activity. Axin2 levels are elevated in Runx2(-/-) calvarial cells, and Runx2 represses transcription of Axin2 mRNA, suggesting a direct relationship between these factors in vivo. Here we demonstrate that Runx2 binds several regions of the Axin2 promoter and that Runx2-mediated repression of Axin2 transcription depends on Hdac3. To determine whether Runx2 contributes to the etiology of Axin2 deficiency-induced craniosynostosis, we generated Axin2(-/-):Runx2(+/-) mice. These double mutant mice had longer skulls than Axin2(-/-) mice, indicating that Runx2 haploinsufficiency rescued the craniosynostosis phenotype of Axin2(-/-) mice. Together, these studies identify a key mechanistic pathway for regulating intramembranous bone development within the skull that involves Runx2- and Hdac3-mediated suppression of Axin2 to prevent the untimely closure of the calvarial sutures.

The ß-catenin destruction complex.

The Wnt/ß-catenin pathway is highly regulated to insure the correct temporal and spatial activation of its target genes. In the absence of a Wnt stimulus, the transcriptional coactivator ß-catenin is degraded by a multiprotein "destruction complex" that includes the tumor suppressors Axin and adenomatous polyposis coli (APC), the Ser/Thr kinases GSK-3 and CK1, protein phosphatase 2A (PP2A), and the E3-ubiquitin ligase ß-TrCP. The complex generates a ß-TrCP recognition site by phosphorylation of a conserved Ser/Thr-rich sequence near the ß-catenin amino terminus, a process that requires scaffolding of the kinases and ß-catenin by Axin. Ubiquitinated ß-catenin is degraded by the proteasome. The molecular mechanisms that underlie several aspects of destruction complex function are poorly understood, particularly the role of APC. Here we review the molecular mechanisms of destruction complex function and discuss several potential roles of APC in ß-catenin destruction.