Roles of Axin in the Wnt signalling pathway

The Wnt signalling pathway is conserved in various species from worms to mammals, and plays important roles in development, cellular proliferation, and differentiation. The molecular mechanisms by which the Wnt signal regulates cellular functions are becoming increasingly well understood. Wnt stabilizes cytoplasmic beta-catenin, which stimulates the expression of genes including c-myc, c-jun, fra-1, and cyclin D1. Axin, newly recognized as a component of the Wnt signalling pathway, negatively regulates this pathway. Other components of the Wnt signalling pathway, including Dvl, glycogen synthase kinase-3beta, beta-catenin, and adenomatous polyposis coli, interact with Axin, and the phosphorylation and stability of beta-catenin are regulated in the Axin complex. Thus, Axin acts as a scaffold protein in the Wnt signalling pathway, thereby regulating cellular functions.     

Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.     

Caveolin is necessary for Wnt-3a-dependent internalization of LRP6 and accumulation of beta-catenin

beta-catenin-mediated Wnt signaling is critical in animal development and tumor progression. The single-span transmembrane Wnt receptor, low-density lipoprotein receptor-related protein 6 (LRP6), interacts with Axin to promote the Wnt-dependent accumulation of beta-catenin. However, the molecular mechanism of receptor internalization and its impact on signaling are unclear. Here, we present evidence that LRP6 is internalized with caveolin and that the components of this endocytic pathway are required not only for Wnt-3a-induced internalization of LRP6 but also for accumulation of beta-catenin. Overall, our data suggest that Wnt-3a triggers the interaction of LRP6 with caveolin and promotes recruitment of Axin to LRP6 phosphorylated by glycogen synthase kinase-3beta and that caveolin thereby inhibits the binding of beta-catenin to Axin. Thus, caveolin plays critical roles in inducing the internalization of LRP6 and activating the Wnt/beta-catenin pathway. We also discuss the idea that distinct endocytic pathways correlate with the specificity of Wnt signaling events.     

Adenomatous polyposis coli is down-regulated by the ubiquitin-proteasome pathway in a process facilitated by Axin

Adenomatous polyposis coli (APC) protein and Axin form a complex that mediates the down-regulation of beta-catenin, a key effector of Wnt signaling. Truncation mutations in APC are responsible for familial and sporadic colorectal tumors due to failure in the down-regulation of beta-catenin. While the regulation of beta-catenin by APC has been extensively studied, the regulation of APC itself has received little attention. Here we show that the level of APC is down-regulated by the ubiquitin-proteasome pathway and that Wnt signaling inhibits the process. The domain responsible for the down-regulation and direct ubiquitination was identified. We also show an unexpected role for Axin in facilitating the ubiquitination-proteasome-mediated down-regulation of APC through the oligomerization of Axin. Our results suggest a new mechanism for the regulation of APC by Axin and Wnt signaling.     

Shoc2/Sur8 Protein Regulates Neurite Outgrowth

The Shoc2 protein has been implicated in the positive regulation of the Ras-ERK pathway by increasing the functional binding interaction between Ras and Raf, leading to increased ERK activity. Here we found that Shoc2 overexpression induced sustained ERK phosphorylation, notably in the case of EGF stimulation, and Shoc2 knockdown inhibited ERK activation. We demonstrate that ectopic overexpression of human Shoc2 in PC12 cells significantly promotes neurite extension in the presence of EGF, a stimulus that induces proliferation rather than differentiation in these cells. Finally, Shoc2 depletion reduces both NGF-induced neurite outgrowth and ERK activation in PC12 cells. Our data indicate that Shoc2 is essential to modulate the Ras-ERK signaling outcome in cell differentiation processes involved in neurite outgrowth.     

Inhibition of Wnt signaling pathway by a novel axin-binding protein

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3beta efficiently phosphorylates beta-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3beta-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta-catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.     

Control of beta-catenin phosphorylation/degradation by a dual-kinase mechanism

Wnt regulation of beta-catenin degradation is essential for development and carcinogenesis. beta-catenin degradation is initiated upon amino-terminal serine/threonine phosphorylation, which is believed to be performed by glycogen synthase kinase-3 (GSK-3) in complex with tumor suppressor proteins Axin and adnomatous polyposis coli (APC). Here we describe another Axin-associated kinase, whose phosphorylation of beta-catenin precedes and is required for subsequent GSK-3 phosphorylation of beta-catenin. This "priming" kinase is casein kinase Ialpha (CKIalpha). Depletion of CKIalpha inhibits beta-catenin phosphorylation and degradation and causes abnormal embryogenesis associated with excessive Wnt/beta-catenin signaling. Our study uncovers distinct roles and steps of beta-catenin phosphorylation, identifies CKIalpha as a component in Wnt/beta-catenin signaling, and has implications to pathogenesis/therapeutics of human cancers and diabetes.     

Axin: a master scaffold for multiple signaling pathways

Axin was originally identified from the characterization of the Fused locus, the disruption of which leads to duplication of axis and embryonic lethality. It is a multidomain protein that interacts with multiple proteins and functions as a negative regulator of Wnt signaling by downregulating the beta-catenin levels. Recently, it was demonstrated that Axin also plays an important role in a JNK signaling pathway. Axin utilizes discriminatory domains for its distinct roles in the Wnt pathway and in the Axin/JNK pathway. Here we review the data that show how Axin regulates multiple signaling pathways by serving as a scaffold protein, controlling diverse cellular functions in proliferation, fate determination, and suppression of tumorigenesis.     

Oncogenic Ras abrogates MEK SUMOylation that suppresses the ERK pathway and cell transformation

The ERK (extracellular signal-regulated kinase) MAPK (mitogen-activated protein kinase) cascade (Raf-MEK-ERK) mediates mitogenic signalling, and is frequently hyperactivated by Ras oncogenes in human cancer. The entire range of activities of multifunctional Ras in carcinogenesis remains elusive. Here we report that the ERK pathway is downregulated by MEK (MAPK-ERK kinase) SUMOylation, which is inhibited by oncogenic Ras. MEK SUMOylation blocked ERK activation by disrupting the specific docking interaction between MEK and ERK. Expression of un-SUMOylatable MEK enhanced ERK activation, cell differentiation, proliferation and malignant transformation by oncogenic ErbB2 or Raf, but not by active Ras. Interestingly, MEK SUMOylation was abrogated in cancer cells harbouring Ras mutations. Oncogenic Ras inhibits MEK SUMOylation by impairing the function of the MEKK1 MAPKKK as a SUMO-E3 ligase specific for MEK. Furthermore, forced enhancement of MEK SUMOylation suppressed Ras-induced cell transformation. Thus, oncogenic Ras efficiently activates the ERK pathway both by activating Raf and by inhibiting MEK SUMOylation, thereby inducing carcinogenesis.     

Phosphorylation of axin, a Wnt signal negative regulator, by glycogen synthase kinase-3beta regulates its stability

Axin forms a complex with glycogen synthase kinase-3beta (GSK-3beta) and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin, thereby stimulating the degradation of beta-catenin. Because GSK-3beta also phosphorylates Axin in the complex, the physiological significance of the phosphorylation of Axin was examined. Treatment of COS cells with LiCl, a GSK-3beta inhibitor, and okadaic acid, a protein phosphatase inhibitor, decreased and increased, respectively, the cellular protein level of Axin. Pulse-chase analyses showed that the phosphorylated form of Axin was more stable than the unphosphorylated form and that an Axin mutant, in which the possible phosphorylation sites for GSK-3beta were mutated, exhibited a shorter half-life than wild type Axin. Dvl-1, which was genetically shown to function upstream of GSK-3beta, inhibited the phosphorylation of Axin by GSK-3beta in vitro. Furthermore, Wnt-3a-containing conditioned medium down-regulated Axin and accumulated beta-catenin in L cells and expression of Dvl-1(DeltaPDZ), in which the PDZ domain was deleted, suppressed this action of Wnt-3a. These results suggest that the phosphorylation of Axin is important for the regulation of its stability and that Wnt down-regulates Axin through Dvl.     

The tuberin-hamartin complex negatively regulates beta-catenin signaling activity

Tuberous sclerosis complex (TSC) is characterized by the formation of hamartomas in multiple organs resulting from mutations in the TSC1 or TSC2 gene. Their protein products, hamartin and tuberin, respectively, form a functional complex that affects cell growth, differentiation, and proliferation. Several lines of evidence, including renal tumors derived from TSC2+/- animals, suggest that the loss or inhibition of tuberin is associated with up-regulation of cyclin D1. As cyclin D1 can be regulated through the canonical Wnt/beta-catenin signaling pathway, we hypothesize that the cell proliferative effects of hamartin and tuberin are partly mediated through beta-catenin. In this study, total beta-catenin protein levels were found to be elevated in the TSC2-related renal tumors. Ectopic expression of hamartin and wild-type tuberin, but not mutant tuberin, reduced beta-catenin steady-state levels and its half-life. The TSC1-TSC2 complex also inhibited Wnt-1 stimulated Tcf/LEF luciferase reporter activity. This inhibition was eliminated by constitutively active beta-catenin but not by Disheveled, suggesting that hamartin and tuberin function at the level of the beta-catenin degradation complex. Indeed, hamartin and tuberin co-immunoprecipitated with glycogen synthase kinase 3 beta and Axin, components of this complex in a Wnt-1-dependent manner. Our data suggest that hamartin and tuberin negatively regulate beta-catenin stability and activity by participating in the beta-catenin degradation complex.     

Differential molecular assemblies underlie the dual function of Axin in modulating the WNT and JNK pathways.

Axin is a multidomain scaffold protein that exerts a dual function in the Wnt signaling and MEKK1/JNK pathways. This raises a critical question as to whether Axin-based differential molecular assemblies exist and how these may act to coordinate the two separate pathways. Here we show that both wild-type glycogen synthase kinase-3 beta (GSK-3 beta) and kinase-dead GSK-3 beta-Y216F (capable of binding to Axin), but not GSK-3 beta-K85M (incapable of binding to Axin in mammalian cells), prevented MEKK1 binding to the Axin complex, thereby inhibiting JNK activation. We further show that casein kinase I epsilon also inhibited Axin-mediated JNK activation by competing against MEKK1 binding. In contrast, beta-catenin and adenomatous polyposis coli binding did not affect MEKK1 binding to the same Axin complex. This suggests that even when Axin is "switched" to activate the JNK pathway, it is still capable of sequestering free beta-catenin, which is a critical aspect for cellular homeostasis. Our results clearly demonstrate that differential molecular assemblies underlie the duality of Axin functions in the negative regulation of Wnt signaling and activation of the JNK MAPK pathway.     

The PI3 kinase-Akt pathway mediates Wnt3a-induced proliferation

Wnt3a activates proliferation of fibroblasts cells via activation of both extracellular signal-regulated kinase (ERK) and Wnt/beta-catenin signaling pathways. In this study, we show that the phosphatidyl inositol 3 kinases (PI3K)-Akt pathway is also involved in the Wnt3a-induced proliferation. Akt was activated within 30 min by Wnt3a in NIH3T3 cells. By Wnt3a treatment, activated Akt was transiently accumulated in nucleus although beta-catenin was accumulated in the nucleus of cells in a prolonged manner. The Wnt3a-induced Akt activation was not affected by siRNA-mediated reduction of beta-catenin, indicating that Wnt3a-induced Akt activation may occur independently of beta-catenin. The Wnt3a-induced Akt activation was abolished by pre-treatment with PI3K inhibitor, LY294002 and Wortmanin, but not by MEK inhibitor, U0126, indicating that Wnt3a activates Akt via PI3K. The growth and proliferation induced by Wnt3a were blocked by treatments of the PI3K inhibitors. Furthermore, Wnt3a-induced proliferation was blocked by Akt siRNA. These results reveal that the PI3K-Akt pathway mediates the Wnt3a-induced growth and proliferation of NIH3T3 cells.     

The role of Axin2 in calvarial morphogenesis and craniosynostosis

Axin1 and its homolog Axin2/conductin/Axil are negative regulators of the canonical Wnt pathway that suppress signal transduction by promoting degradation of beta-catenin. Mice with deletion of Axin1 exhibit defects in axis determination and brain patterning during early embryonic development. We show that Axin2 is expressed in the osteogenic fronts and periosteum of developing sutures during skull morphogenesis. Targeted disruption of Axin2 in mice induces malformations of skull structures, a phenotype resembling craniosynostosis in humans. In the mutants, premature fusion of cranial sutures occurs at early postnatal stages. To elucidate the mechanism of craniosynostosis, we studied intramembranous ossification in Axin2-null mice. The calvarial osteoblast development is significantly affected by the Axin2 mutation. The Axin2 mutant displays enhanced expansion of osteoprogenitors, accelerated ossification, stimulated expression of osteogenic markers and increases in mineralization. Inactivation of Axin2 promotes osteoblast proliferation and differentiation in vivo and in vitro. Furthermore, as the mammalian skull is formed from cranial skeletogenic mesenchyme, which is derived from mesoderm and neural crest, our data argue for a region-specific effect of Axin2 on neural crest dependent skeletogenesis. The craniofacial anomalies caused by the Axin2 mutation are mediated through activation of beta-catenin signaling, suggesting a novel role for the Wnt pathway in skull morphogenesis.