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.     

Inhibition of the Wnt signaling pathway by the PR61 subunit of protein phosphatase 2A

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3beta-dependent phosphorylation in the complex and the stability of beta-catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61beta and -gamma, interact with Axin. PR61beta or -gamma formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61beta on Axin was different from those of GSK-3beta, beta-catenin, APC, and Dvl. Although PR61beta did not affect the stability of beta-catenin, it inhibited Dvl- and beta-catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed beta-catenin-induced axis formation and expression of siamois, a Wnt target gene, in Xenopus embryos, suggesting that PR61beta acts either at the level of beta-catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of beta-catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps.     

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin.

Glycogen synthase kinase-3 (GSK-3) mediates epidermal growth factor, insulin and Wnt signals to various downstream events such as glycogen metabolism, gene expression, proliferation and differentiation. We have isolated here a GSK-3beta-interacting protein from a rat brain cDNA library using a yeast two-hybrid method. This protein consists of 832 amino acids and possesses Regulators of G protein Signaling (RGS) and dishevelled (Dsh) homologous domains in its N- and C-terminal regions, respectively. The predicted amino acid sequence of this GSK-3beta-interacting protein shows 94% identity with mouse Axin, which recently has been identified as a negative regulator of the Wnt signaling pathway; therefore, we termed this protein rAxin (rat Axin). rAxin interacted directly with, and was phosphorylated by, GSK-3beta. rAxin also interacted directly with the armadillo repeats of beta-catenin. The binding site of rAxin for GSK-3beta was distinct from the beta-catenin-binding site, and these three proteins formed a ternary complex. Furthermore, rAxin promoted GSK-3beta-dependent phosphorylation of beta-catenin. These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK-3beta and beta-catenin and mediating the signal from GSK-3beta to beta-catenin.     

Regulation of dishevelled and beta-catenin in rat skeletal muscle: an alternative exercise-induced GSK-3beta signaling pathway

beta-catenin is a multifunctional protein involved in cell-cell adhesion and the Wnt signaling pathway. beta-Catenin is activated upon its dephosphorylation, an event triggered by Dishevelled (Dvl)-mediated phosphorylation and deactivation of glycogen synthase kinase-3beta (GSK-3beta). In skeletal muscle, both insulin and exercise decrease GSK-3beta activity, and we tested the hypothesis that these two stimuli regulate beta-catenin. Immunoblotting demonstrated that Dvl, Axin, GSK-3beta, and beta-catenin proteins are expressed in rat red and white gastrocnemius muscles. Treadmill running exercise in vivo significantly decreased beta-catenin phosphorylation in both muscle types, with complete dephosphorylation being elicited by maximal exercise. beta-Catenin dephosphorylation was intensity dependent, as dephosphorylation was highly correlated with muscle glycogen depletion during exercise (r(2) = 0.84, P < 0.001). beta-Catenin dephosphorylation was accompanied by increases in GSK-3beta Ser(9) phosphorylation and Dvl-GSK-3beta association. In contrast to exercise, maximal insulin treatment (1 U/kg body wt) had no effect on skeletal muscle beta-catenin phosphorylation or Dvl-GSK-3beta interaction. In conclusion, exercise in vivo, but not insulin, increases the association between Dvl and GSK-3beta in skeletal muscle, an event paralleled by beta-catenin dephosphorylation.     

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.     

DIX Domains of Dvl and Axin Are Necessary for Protein Interactions and Their Ability To Regulate β-Catenin Stability

The N-terminal region of Dvl-1 (a mammalian Dishevelled homolog) shares 37% identity with the C-terminal region of Axin, and this related region is named the DIX domain. The functions of the DIX domains of Dvl-1 and Axin were investigated. By yeast two-hybrid screening, the DIX domain of Dvl-1 was found to interact with Dvl-3, a second mammalian Dishevelled relative. The DIX domains of Dvl-1 and Dvl-3 directly bound one another. Furthermore, Dvl-1 formed a homo-oligomer. Axin also formed a homo-oligomer, and its DIX domain was necessary. The N-terminal region of Dvl-1, including its DIX domain, bound to Axin directly. Dvl-1 inhibited Axin-promoted glycogen synthase kinase 3beta-dependent phosphorylation of beta-catenin, and the DIX domain of Dvl-1 was required for this inhibitory activity. Expression of Dvl-1 in L cells induced the nuclear accumulation of beta-catenin, and deletion of the DIX domain abolished this activity. Although expression of Axin in SW480 cells caused the degradation of beta-catenin and reduced the cell growth rate, expression of an Axin mutant that lacks the DIX domain did not affect the level of beta-catenin or the growth rate. These results indicate that the DIX domains of Dvl-1 and Axin are important for protein-protein interactions and that they are necessary for the ability of Dvl-1 and Axin to regulate the stability of beta-catenin.     

Axin directly interacts with plakoglobin and regulates its stability.

Plakoglobin is homologous to beta-catenin. Axin, a Wnt signal negative regulator, enhances glycogen synthase kinase (GSK)-3beta-dependent phosphorylation of beta-catenin and stimulates the degradation of beta-catenin. Therefore, we examined the effect of Axin on plakoglobin stability. Axin formed a complex with plakoglobin in COS cells and SW480 cells. Axin directly bound to plakoglobin, and this binding was inhibited by beta-catenin. Axin promoted GSK-3beta-dependent phosphorylation of plakoglobin. Furthermore, overexpression of Axin down-regulated the level of plakoglobin in SW480 cells. These results suggest that Axin regulates the stability of plakoglobin by enhancing its phosphorylation by GSK-3beta and that Axin may act on beta-catenin and plakoglobin in similar manners.     

Targeted degradation of beta-catenin by chimeric F-box fusion proteins

Adenomatous polyposis coli (APC) tumor suppressor protein, together with Axin and glycogen synthase kinase 3beta (GSK-3beta), forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of cytoplasmic beta-catenin by ubiquitin-dependent proteolysis. Degradation of phosphorylated beta-catenin is initiated by interaction through the WD40-repeat of a F-box protein beta-TrCP, a component of SCF ubiquitin ligase complex. Mutations in APC, Axin, and beta-catenin that prevent down-regulation of cytoplasmic beta-catenin are found in various types of cancers. In the search for efficient treatment and prevention of malignancies associated with increased levels of cytoplasmic beta-catenin, we created chimeric F-box fusion proteins by replacing the WD40-repeat of beta-TrCP with the beta-catenin-binding domains of Tcf4 and E-cadherin. Expression of chimeric F-box fusion proteins successfully promotes degradation of beta-catenin independently of GSK-3beta-mediated phosphorylation. More importantly, this degradation does not require intact APC protein (pAPC).     

An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin

beta-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of beta-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. beta-catenin levels are regulated by the ubiquitin-dependent proteolysis system and beta-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3beta (GSK-3beta)/Axin kinase complex. Here we show that FWD1 (the mouse homologue of Slimb/betaTrCP), an F-box/WD40-repeat protein, specifically formed a multi-molecular complex with beta-catenin, Axin, GSK-3beta and APC. Mutations at the signal-induced phosphorylation site of beta-catenin inhibited its association with FWD1. FWD1 facilitated ubiquitination and promoted degradation of beta-catenin, resulting in reduced cytoplasmic beta-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibited the ubiquitination process and stabilized beta-catenin. These results suggest that the Skp1/Cullin/F-box protein FWD1 (SCFFWD1)-ubiquitin ligase complex is involved in beta-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated beta-catenin. FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of beta-catenin in response to external signals. SCFFWD1 may be critical for tumor development and suppression through regulation of beta-catenin protein stability.     

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.     

Complex formation of adenomatous polyposis coli gene product and axin facilitates glycogen synthase kinase-3 beta-dependent phosphorylation of beta-catenin and down-regulates beta-catenin

Adenomatous polyposis coli gene product (APC) functions as a tumor suppressor and its mutations in familial adenomatous polyposis and colorectal cancers lead to the accumulation of cytoplasmic beta-catenin. The molecular mechanism by which APC regulates the stability of beta-catenin was investigated. The central region of APC, APC-(1211-2075), has the beta-catenin- and Axin-binding sites and down-regulates beta-catenin. Glycogen synthase kinase-3 beta (GSK-3 beta) phosphorylated beta-catenin slightly in the presence of either APC-(1211-2075) or Axin(delta)(beta)(-catenin), in which the beta-catenin-binding site is deleted, and greatly in the presence of both proteins. The enhancement of the GSK-3 beta-dependent phosphorylation of beta-catenin was eliminated by the APC-binding site of Axin. Axin down-regulated beta-catenin in SW480 cells, but not Axin(delta)(beta)(-catenin). In L cells where APC is intact, Axin(delta)(beta)(-catenin) inhibited Wnt-dependent accumulation of beta-catenin but not Axin-(298-832)(delta)(beta)(-catenin) in which the APC- and beta-catenin-binding sites are deleted. These results indicate that the complex formation of APC and Axin enhances the phosphorylation of beta-catenin by GSK-3 beta, leading to the down-regulation of beta-catenin.