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.     

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.     

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.     

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.     

Expression and characterization of GSK-3 mutants and their effect on beta-catenin phosphorylation in intact cells

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates beta-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of beta-catenin by GSK-3 does not require priming in vitro, i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3beta. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7, 1321-1327). Here we have investigated whether phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin requires priming through prior phosphorylation at Ser(45) in intact cells. We have shown that the Arg(96) mutant does not induce beta-catenin degradation but instead stabilizes beta-catenin, indicating that it is unable to phosphorylate beta-catenin in intact cells. Furthermore, if Ser(45) in beta-catenin is mutated to Ala, beta-catenin is markedly stabilized, and phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin by wild type GSK-3beta is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro, is still able to phosphorylate beta-catenin in intact cells although it has reduced activity. Mutation of Tyr(216) to Phe markedly reduces the ability of GSK-3beta to phosphorylate and down-regulate beta-catenin. In conclusion, we have found that the Arg(96) mutant has a dominant-negative effect on GSK-3beta-dependent phosphorylation of beta-catenin and that targeting of beta-catenin for degradation requires prior priming through phosphorylation of Ser(45).     

The structure of phosphorylated GSK-3beta complexed with a peptide, FRATtide, that inhibits beta-catenin phosphorylation.

BACKGROUND: Glycogen synthase kinase-3 (GSK-3) sequentially phosphorylates four serine residues on glycogen synthase (GS), in the sequence SxxxSxxxSxxx-SxxxS(p), by recognizing and phosphorylating the first serine in the sequence motif SxxxS(P) (where S(p) represents a phosphoserine). FRATtide (a peptide derived from a GSK-3 binding protein) binds to GSK-3 and blocks GSK-3 from interacting with Axin. This inhibits the Axin-dependent phosphorylation of beta-catenin by GSK-3. RESULTS: Structures of uncomplexed Tyr216 phosphorylated GSK-3beta and of its complex with a peptide and a sulfate ion both show the activation loop adopting a conformation similar to that in the phosphorylated and active forms of the related kinases CDK2 and ERK2. The sulfate ion, adjacent to Val214 on the activation loop, represents the binding site for the phosphoserine residue on 'primed' substrates. The peptide FRATtide forms a helix-turn-helix motif in binding to the C-terminal lobe of the kinase domain; the FRATtide binding site is close to, but does not obstruct, the substrate binding channel of GSK-3. FRATtide (and FRAT1) does not inhibit the activity of GSK-3 toward GS. CONCLUSIONS: The Axin binding site on GSK-3 presumably overlaps with that for FRATtide; its proximity to the active site explains how Axin may act as a scaffold protein promoting beta-catenin phosphorylation. Tyrosine 216 phosphorylation can induce an active conformation in the activation loop. Pre-phosphorylated substrate peptides can be modeled into the active site of the enzyme, with the P1 residue occupying a pocket partially formed by phosphotyrosine 216 and the P4 phosphoserine occupying the 'primed' binding site.     

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.     

Purification of GSK-3 by affinity chromatography on immobilized axin

Glycogen synthase kinase 3 (GSK-3), an element of the Wnt signalling pathway, plays a key role in numerous cellular processes including cell proliferation, embryonic development, and neuronal functions. It is directly involved in diseases such as cancer (by controlling apoptosis and the levels of beta-catenin and cyclin D1), Alzheimer's disease (tau hyperphosphorylation), and diabetes (as a downstream element of insulin action, GSK-3 regulates glycogen and lipid synthesis). We describe here a rapid and efficient method for the purification of GSK-3 by affinity chromatography on an immobilized fragment of axin. Axin is a docking protein which interacts with GSK-3ss, beta-catenin, phosphatase 2A, and APC. A polyhistidine-tagged axin peptide (residues 419-672) was produced in Escherichia coli and either immobilized on Ni-NTA agarose beads or purified and immobilized on CNBr-activated Sepharose 4B. These "Axin-His6" matrices were found to selectively bind recombinant rat GSK-3 beta and native GSK-3 from yeast, sea urchin embryos, and porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to follow the status of GSK-3 (protein level, phosphorylation state, kinase activity) under various physiological settings. It also provides a simple and efficient way to purify large amounts of active recombinant or native GSK-3 for screening purposes.     

Characterisation of the phosphorylation of beta-catenin at the GSK-3 priming site Ser45

Activation of the canonical Wnt signalling pathway results in stabilisation and nuclear translocation of beta-catenin. In the absence of a Wnt signal, beta-catenin is phosphorylated at four conserved serine and threonine residues at the N-terminus of the protein, which results in beta-catenin ubiquitination and proteasome-dependent degradation. The phosphorylation of three of these residues, Thr41, Ser37, and Ser33, is mediated by glycogen synthase kinase-3 (GSK-3) in a sequential manner, beginning from the C-terminal Thr41. It has recently been shown that the GSK-3 dependent phosphorylation of beta-catenin requires prior priming through phosphorylation of Ser45. However, it is not known whether phosphorylation of Ser45 is carried out by GSK-3 itself or by an alternative kinase. In this study, the phosphorylation of beta-catenin at Ser45 was characterised using a phospho-specific antibody. GSK-3beta was found to be unable to phosphorylate beta-catenin at Ser45 in vitro and in intact cells. However, inhibition of GSK-3 in intact cells reduced Ser45 phosphorylation, suggesting that GSK-3 kinase activity is required for the phosphorylation event. In vitro, CK1, but not CK2, phosphorylates Ser45. Ser45 phosphorylation in intact cells is not mediated by CK1varepsilon, a known positive regulator of Wnt signalling, as overexpression of this kinase leads to decreased phosphorylation levels. In conclusion, phosphorylation of beta-catenin at the GSK-3 priming site Ser45 is not mediated by GSK-3 itself, but by an alternative kinase, indicating that beta-catenin is not an unprimed substrate for GSK-3 in vivo. Priming of GSK-3 dependent phosphorylation of beta-catenin by a different kinase could have important implications for the regulation of Wnt signalling.     

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.     

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.     

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.