β-Catenin, a member of the Wnt signaling pathway, is downregulated by glycogen synthase kinase-3β (GSK-3β)-dependent phosphorylation of Ser/Thr residues in the N-terminus of the protein, followed by ubiquitination and proteosomal degradation. In human and rodent cancers, mutations that substitute one of the critical Ser/Thr residues in the GSK-3β region of β-catenin stabilize the protein and activate β-catenin/TCF/LEF target genes. This study examined three oncogenic β-catenin mutants from rat colon tumors containing substitutions adjacent to amino-acid residue Ser33, a key target for phosphorylation by GSK-3β. Compared with wild-type β-catenin (WT), the β-catenin mutants D32G, D32N, and D32Y strongly activated TCF-4-dependent transcription in HEK293 cells, and there was accumulation of β-catenin in the cell lysates. Immunoblotting with phosphospecific antibodies indicated that there was little if any effect on the phosphorylation of Ser37, Thr41 or Ser45; however, the phosphorylation of Ser33 appeared to be affected in the β-catenin mutants. Specifically, antiphospho-β-catenin 33/37/41 antibody identified high, intermediate and low expression levels of phosphorylated β-catenin in cells transfected with D32G, D32N and D32Y, respectively. Experiments with the proteosome inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN) revealed ubiquitinated bands on all three mutant β-catenins, as well as on WT β-catenin. The relative order of ubiquitination was WT > D32G > D32N > D32Y, in parallel with findings from the phosphorylation studies. These results are discussed in the context of previous studies, which indicated that amino-acid residue D32 lies within the ubiquitination recognition motif of β-catenin.
β-catenin; TCF/LEF; APC; Wnt signaling; colorectal cancer; CTNNB1; human β-catenin gene; Ctnnb1; rat β-catenin gene
The effector protein of the canonical Wnt pathway is β-catenin, which is regulated by the ubiquitin system. This study shows that the E3 ubiquitin ligase EDD ubiquitinates β-catenin, leading to up-regulation of β-catenin's expression levels and activity. Thus the results demonstrate a role for the ubiquitin system in up-regulation of the Wnt pathway.
Wnt/β-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. β-Catenin activity is tightly regulated via a multiprotein complex that includes the kinase glycogen synthase kinase-3β (GSK-3β). GSK-3β phosphorylates β-catenin, marking it for ubiquitination and degradation via the proteasome. Thus in regulation of the Wnt pathway, the ubiquitin system is known to be involved mostly in mediating the turnover of β-catenin, resulting in reduced Wnt signaling levels. Here we report that an arm of the ubiquitin system increases β-catenin protein levels. We show that GSK-3β directly interacts with the E3 ubiquitin ligase identified by differential display (EDD) that also binds β-catenin. Expression of EDD leads to enhanced nuclear accumulation of both GSK-3β and β-catenin and results in up-regulation of β-catenin expression levels and activity. Importantly, EDD ubiquitinates β-catenin through Lys29- or Lys11-linked ubiquitin chains, leading to enhanced stability of β-catenin. Our results demonstrate a role for the ubiquitin system in up-regulation of the Wnt signaling pathway, suggesting that EDD could function as a colorectal oncogene.
The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease.
Beta-catenin can be cleaved by caspase-3 or degraded by activated glycogen synthase kinase-3β via phosphorylating β-catenin. We tested the hypothesis that β-catenin undergoes degradation after stroke, and its degradation is dependent on caspase activity. Stroke was generated by permanent middle cerebral artery occlusion and 1h of transient bilateral common carotid artery occlusion in rats. Active caspase-3 was expressed in the ischemic cortex from 5 to 48 h after stroke, whereas β-catenin markedly degraded at 24 and 48 h after stroke. The caspase 3-specific inhibitor, Z-DQMD-FMK, attenuated β-catenin degradation, but it did not affect phosphorylation of both β-catenin and glycogen synthase kinase-3β. In conclusion, β-catenin degraded after stroke, and its degradation was caspase-3 dependent.
β-catenin; caspase-3; focal ischemia; glycogen synthase kinase-3β; stroke
Traumatic brain injury (TBI) sets in motion cascades of biochemical changes that result in delayed cell death and altered neuronal architecture. Studies have demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3) effectively reduces apoptosis following a number of stimuli. The Wnt family of proteins, and growth factors are two major factors that regulate GSK-3 activity. In the absence of stimuli, GSK-3 is constitutively active and is complexed with Axin, adenomatous polyposis coli (APC), and casein kinase Iα (CK1α) and phosphorylates ß-Catenin leading to its degradation. Binding of Wnt to Frizzled receptors causes the translocation of GSK-3 to the plasma membrane, where it phosphorylates and inactivates the Frizzled co-receptor lipoprotein-related protein 6 (LRP6). Furthermore, the translocation of GSK-3 reduces ß-Catenin phosphorylation and degradation, leading to ß-Catenin accumulation and gene expression. Growth factors activate Akt, which in turn inhibits GSK-3 activity by direct phosphorylation, leading to a reduction in apoptosis.
Using a rodent model, we found that TBI caused a rapid, but transient, increase in LRP6 phosphorylation that is followed by a modest decrease in ß-Catenin phosphorylation. Phospho-GSK-3β immunoreactivity was found to increase three days post injury, a time point at which increased Akt activity following TBI has been observed. Lithium influences several neurochemical cascades, including inhibiting GSK-3. When the efficacy of daily lithium was assessed, reduced hippocampal neuronal cell loss and learning and memory improvements were observed. These influences were partially mimicked by administration of the GSK-3-selective inhibitor SB-216763, as this drug resulted in improved motor function, but only a modest improvement in memory retention and no overt neuroprotection.
Taken together, our findings suggest that selective inhibition of GSK-3 may offer partial cognitive improvement. As a broad spectrum inhibitor of GSK-3, lithium offers neuroprotection and robust cognitive improvement, supporting its clinical testing as a treatment for TBI.
Dishevelled (DVL) is associated with axonal microtubules and regulates microtubule stability through the inhibition of the serine/threonine kinase, glycogen synthase kinase 3β (GSK-3β). In the canonical WNT pathway, the negative regulator Axin forms a complex with β-catenin and GSK-3β, resulting in β-catenin degradation. Inhibition of GSK-3β by DVL increases β-catenin stability and TCF transcriptional activation. Here, we show that Axin associates with microtubules and unexpectedly stabilizes microtubules through DVL. In turn, DVL stabilizes microtubules by inhibiting GSK-3β through a transcription- and β-catenin–independent pathway. More importantly, axonal microtubules are stabilized after DVL localizes to axons. Increased microtubule stability is correlated with a decrease in GSK-3β–mediated phosphorylation of MAP-1B. We propose a model in which Axin, through DVL, stabilizes microtubules by inhibiting a pool of GSK-3β, resulting in local changes in the phosphorylation of cellular targets. Our data indicate a bifurcation in the so-called canonical WNT-signaling pathway to regulate microtubule stability.
β-catenin; GSK-3β; neurons; cytoskeleton; axin
Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is β-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 β-catenin form a complex that promotes the GSK-3–mediated phosphorylation and subsequent degradation of β-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates β-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 β-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 β-catenin levels and establishment of the dorsal axis.
Wnt pathway; dorsal/ventral; β-catenin
Glycogen synthase kinase 3β (GSK3β) is involved in metabolism, neurodegeneration, and cancer. Inhibition of GSK3β activity is the primary mechanism that regulates this widely expressed active kinase. Although the protein kinase Akt inhibits GSK3β by phosphorylation at the N terminus, preventing Akt-mediated phosphorylation does not affect the cell-survival pathway activated through the GSK3β substrate β-catenin. Here, we show that p38 mitogen-activated protein kinase (MAPK) also inactivates GSK3β by direct phosphorylation at its C terminus, and this inactivation can lead to an accumulation of β-catenin. p38 MAPK-mediated phosphorylation of GSK3β occurs primarily in the brain and thymocytes. Activation of β-catenin-mediated signaling through GSK3β inhibition provides a potential mechanism for p38 MAPK-mediated survival in specific tissues.
The latency-associated nuclear antigen (LANA) of Karposi's sarcoma-associated herpesvirus has been reported to interact with glycogen synthase kinase 3β (GSK-3β) and regulate its activity, leading to inhibition of GSK-3-dependent β-catenin degradation. In this study, the interaction between LANA and GSK-3β was characterized further. LANA was found to interact with GSK-3β in vitro as well as in intact cells. However, LANA did not regulate GSK-3β kinase activity and LANA-induced upregulation of β-catenin was GSK-3β independent. LANA did not regulate the stability of β-catenin or of its reported interaction partners p53 and von Hippel-Lindau protein. Additional targets of LANA are likely to mediate its malignancy-promoting function.
Aims: Activation of specific signaling pathways in response to mechanical trauma causes delayed neuronal apoptosis; GSK-3β/β-catenin signaling plays a critical role in the apoptosis of neurons in CNS diseases, SGK was discovered as a regulator of GSK-3β/β-catenin pathway, The goal of this study was to determine if the mechanism of cell death or survival mediated by the SGK/GSK-3β/β-catenin pathway is involved in a rat model of TBI. Main methods: Here, an acute traumatic brain injury model was applied to investigate the expression change and possible roles of SGK, Expression of SGK, and total-GSK-3β, phospho-GSK3β on ser-9, beta-catenin, and caspase-3 were examined by immunohistochemistry and Western blot analysis. Double immunofluorescent staining was used to observe the SGK localizations. Si-RNA was performed to identify whether SGK regulates neuron apoptosis via GSK-3β/β-catenin pathway, ultimately inhibit caspase-3 activation. Key findings: Temporally, SGK expression showed an increase pattern after TBI and reached a peak at day 3. Spatially, SGK was widely expressed in the neuron, rarely in astrocytes and oligodendrocytes; in addition, the expression patterns of active caspase-3 and phospho-GSK3β were parallel with that of SGK, at the same time, the expression of β-catenin shows similarity with SGK. In vitro, to further investigate the function of SGK, a neuronal cell line PC12 was employed to establish an apoptosis model. We analyzed the association of SGK with apoptosis on PC12 cells by western blot, immunofluorescent labeling and siRNA. Significance: the results implied that SGK plays an important role in neuron apoptosis via the regulation of GSK3β/β-catenin signaling pathway; ultimately inhibit caspase-3 activation. Taken together, we inferred traumatic brain injury induced an upregulation of SGK in the central nervous system, which show a protective role in neuron apoptosis.
SGK; GSK3β/β-catenin signaling pathway; traumatic brain injury (TBI); neuron apoptosis
Increasing evidence shows that there is an interaction between mitogen-activated protein kinase and Wnt signaling and that their interaction plays important roles in a variety of cellular processes. However, how the two signaling interacts is not clear. In this study, we found that β-catenin expression was strikingly increased in the intestinal normal mucosa and tumors of c-Jun N-terminal kinase (JNK) 1-deficient mice by immunohistochemical staining and that both β-catenin expression and transcriptional activity were significantly upregulated in JNK1-deficient mouse embryonic fibroblasts. However, active JNK1 significantly inhibited β-catenin expression and suppressed β-catenin-mediated transcriptional activity by enhancing glycogen synthase kinase 3β (GSK3β) activity. But β-catenin inhibition was significantly reduced by GSK3β RNA interference or GSK3β inhibitor lithium chloride and proteasome inhibitor MG132. Further, mutant β-catenin at the phosphorylation sites of Ser33 and Ser37 by GSK3β was resistant to activated JNK1-induced β-catenin degradation. Moreover, the physical interaction between JNK1 and β-catenin was detected by immunoprecipitation, and their colocalization was seen in cellular nuclei and cytoplasm. Taken together, our data provide direct evidence that JNK1 interacts with and negatively regulates β-catenin signaling through GSK3β pathway and that the β-catenin alteration is probably responsible for the intestinal tumor formation in JNK1-deficient mice.
Hypothermia is robustly protective in pre-clinical models of both global and focal ischemia, as well as in patients after cardiac arrest. Although the mechanism for hypothermic neuroprotection remains unknown, reducing metabolic drive may play a role. Capitalizing on the beneficial effects of hypothermia while avoiding detrimental effects such as infection will be the key to moving this therapy forward as a treatment for stroke. AMPK is a master energy sensor that monitors levels of key energy metabolites. AMPK is activated via phosphorylation (pAMPK) when cellular energy levels are low, such as that seen during ischemia. AMPK activation appears to be detrimental in experimental stroke, likely via exacerbating ischemia-induced metabolic failure. We tested the hypothesis that hypothermia reduces AMPK activation. First, it was found that hypothermia reduced infarct after middle cerebral artery occlusion. Second, induced hypothermia reduced brain pAMPK in both sham control and stroke mice. Third, hypothermic neuroprotection was ameliorated after administration of compound C, an AMPK inhibitor. Finally, deletion of one of the catalytic isoforms of AMPK completely reversed the effect of hypothermia on stroke outcome after both acute and chronic survival. These effects were mediated by a reduction in AMPK activation rather than a reduction in LKB1, an upstream AMPK kinase. In summary, these studies provide evidence that hypothermia exerts its protective effect in part by inhibiting AMPK activation in experimental focal stroke. This suggests that AMPK represents a potentially important biological target for stroke treatment.
AMPK; hypothermia; middle cerebral artery occlusion; stroke
Lineage allocation of the marrow mesenchymal stem cells (MSCs) to osteoblasts and adipocytes is dependent on both Wnt signaling and PPARγ2 activity. Activation of PPARγ2, an essential regulator of energy metabolism and insulin sensitivity, stimulates adipocyte and suppresses osteoblast differentiation and bone formation, and correlates with decreased bone mass and increased fracture rate. In contrast, activation of Wnt signaling promotes osteoblast differentiation, augments bone accrual and reduces total body fat. This study examined the cross-talk between PPARγ2 and β-catenin, a key mediator of canonical Wnt signaling, on MSC lineage determination. Rosiglitazone-activated PPARγ2 induced rapid proteolytic degradation of β-catenin, which was prevented by either inhibiting glycogen synthase kinase 3 beta (GSK3β) activity, or blocking pro-adipocytic activity of PPARγ2 using selective antagonist GW9662 or mutation within PPARγ2 protein. Stabilization of β-catenin suppressed PPARγ2 pro-adipocytic but not anti-osteoblastic activity. Moreover, β-catenin stabilization decreased PPARγ2-mediated insulin signaling as measured by insulin receptor and FoxO1 gene expression, and protein levels of phosphorylated Akt (pAkt). Cellular knockdown of β-catenin with siRNA increased expression of adipocyte but did not affect osteoblast gene markers. Interestingly, the expression of Wnt10b was suppressed by anti-osteoblastic, but not by pro-adipocytic activity of PPARγ2. Moreover, β-catenin stabilization in the presence of activated PPARγ2 did not restore Wnt10b expression indicating a dominant role of PPARγ2 in negative regulation of pro-osteoblastic activity of Wnt signaling. In conclusion, β-catenin and PPARγ2 are in cross-talk which results in sequestration of pro-adipocytic and insulin sensitizing activity. The anti-osteoblastic activity of PPARγ2 is independent of this interaction.
Glycogen synthase kinase-3 (GSK3) plays important roles in numerous signaling pathways that regulate a variety of cellular processes including cell proliferation, differentiation, apoptosis and embryonic development. In the canonical Wnt signaling pathway, GSK3 phosphorylation mediates proteasomal targeting and degradation of β-catenin via the destruction complex. We recently reported a biochemical screen that discovered multiple additional protein substrates whose stability is regulated by Wnt signaling and/or GSK3 and these have important implications for Wnt/GSK3 regulation of different cellular processes.1 In this article, we also present a bio-informatics based screen for proteins whose stability may be controlled by GSK3 and β-Trcp, the SCF E3 ubiquitin ligase that is responsible for β-catenin degradation in the Wnt signaling pathway. Furthermore, we review various GSK3 regulated proteolysis substrates described in the literature. We propose that GSK3 phosphorylation dependent proteolysis is a widespread mechanism that the cell employs to regulate a variety of cell processes in response to signals.
GSK3; wnt; degradation; stability; ubiquitination; proteolysis
Dysregulation of β-catenin turnover due to mutations of its regulatory proteins including APC and p53 is implicated in the pathogenesis of cancer. Thus, intensive effort is being made to search for alternative approaches to reduce abnormally activated β-catenin in cancer cells. Nur77, an orphan member of the nuclear receptor superfamily, plays a role in the growth and apoptosis of cancer cells. Here, we reported that Nur77 could inhibit transcriptional activity of β-catenin by inducing β-catenin degradation via proteasomal degradation pathway that is GSK3β and Siah-1 independent. Nur77 induction of β-catenin degradation required both the N-terminal region of Nur77, which was involved in Nur77 ubiquitination, and the C-terminal region, which was responsible for β-catenin binding. Nur77/ΔDBD, a Nur77 mutant lacking its DNA-binding domain, resided in the cytoplasm, interacted with β-catenin, and induced β-catenin degradation, demonstrating that Nur77-mediated β-catenin degradation was independent of its DNA-binding and transactivation and might occur in the cytoplasm. In addition, we reported our identification of two digitalis-like compounds (DLCs), H-9 and ATE-i2-b4, which potently induced Nur77 expression and β-catenin degradation in SW620 colon cancer cells expressing mutant APC protein in vitro and in animals. DLC-induced Nur77 protein was mainly found in the cytoplasm, and inhibition of Nur77 nuclear export by the CRM1-dependent nuclear export inhibitor leptomycin B or Jun N-terminal kinase inhibitor prevented the effect of DLC on inducing β-catenin degradation. Together, our results demonstrate that β-catenin can be degraded by cytoplasmic Nur77 through their interaction and identify H-9 and ATE-i2-b4 as potent activators of the Nur77-mediated pathway for β-catenin degradation.
Nur77; β-catenin; Nongenomic; Cardenolide; Cancer
Beta-catenin is a multifunctional oncogenic protein that contributes fundamentally to cell development and biology. Elevation in expression and activity of β-catenin has been implicated in many cancers and associated with poor prognosis. Beta-catenin is degraded in the cytoplasm by glycogen synthase kinase 3 beta (GSK-3β) through phosphorylation. Cell growth and proliferation is associated with β-catenin translocation from the cytoplasm into the nucleus.
This laboratory was the first to demonstrate that selenium-containing compounds can enhance the efficacy and cytotoxicity of anticancer drugs in several preclinical xenograft models. These data provided the basis to identify mechanism of selenium action focusing on β-catenin as a target. This study was designed to: (1) determine whether pharmacological doses of methylseleninic acid (MSeA) have inhibitory effects on the level and the oncogenic activity of β-catenin, (2) investigate the kinetics and the mechanism of β-catenin inhibition, and (3) confirm that inhibition of β-catenin would lead to enhanced cytotoxicity of standard chemotherapeutic drugs.
In six human cancer cell lines, the inhibition of total and nuclear expression of β-catenin by MSeA was dose and time dependent. The involvement of GSK-3β in the degradation of β-catenin was cell type dependent (GSK-3β-dependent in HT-29, whereas GSK-3β-independent in HCT-8). However, the pronounced inhibition of β-catenin by MSeA was independent of various drug treatments and was not reversed after combination therapy.
Knockout of β-catenin by ShRNA and its inhibition by MSeA yielded similar enhancement of cytotoxicity of anticancer drugs.
Collectively, the generated data demonstrate that β-catenin is a target of MSeA and its inhibition resulted in enhanced cytotoxicity of chemotherapeutic drugs.
This study demonstrates that β-catenin, a molecule associated with drug resistance, is a target of selenium and its inhibition is associated with increased multiple drugs cytotoxicity in various human cancers. Further, degradation of β-catenin by GSK-3β is not a general mechanism but is cell type dependent.
Modification of the Small Ubiquitin-like Modifier (SUMO) (SUMOylation) appears to regulate diverse cellular processes, including nuclear transport, signal transduction, apoptosis, autophagy, cell cycle control, ubiquitin-dependent degradation and gene transcription. Glycogen synthase kinase 3β (GSK 3β) is a serine/threonine kinase that is thought to contribute to a variety of biological events, including embryonic development, metabolism, tumorigenesis, and cell death. GSK 3β is a constitutively active kinase that regulates many intracellular signaling pathways by phosphorylating substrates such as β-catenin. We noticed that the putative SUMOylation sites are localized on K292 residueof 291FKFPQ295 in GSK 3β based on analysis of the SUMOylation consensus sequence. In this report, we showed that the SUMOylation of GSK 3β occurs on its K292 residue, and this modification promotes its nuclear localization in COS-1. Additionally, our data showed that the GSK 3β SUMO mutant (K292R) decreased its kinase activity and protein stability, affecting cell death. Therefore, our observations at first time suggested that SUMOylation on the K292 residue of GSK 3β might be a GSK 3β regulation mechanism for its kinase activation, subcellular localization, protein stability, and cell apoptosis.
GSK 3β; SUMOylation; cell apoptosis; protein stability; subcellular localization; kinase activation
BACKGROUND: Mutations in the presenilin (PSEN) genes are responsible for the majority of early-onset Alzheimer disease (AD) cases. PSEN1 is a component of a high molecular weight, endoplasmic reticulum, membrane-bound protein complex, including beta-catenin. Pathogenic PSEN1 mutations were demonstrated to have an effect on beta-catenin and glycogen synthase kinase-3beta(GSK-3beta), two members of the wingless Wnt pathway. The nuclear translocation and the stability of beta-catenin, and the interaction between GSK3beta and PSEN1 were influenced. MATERIALS AND METHODS: Stably transfected human embryonic kidney (HEK) 293 cells overexpressing wild-type (wt) and mutant (mt) PSEN1, treated with and without LiCl, were used to isolate cytoplasmic and nuclear fractions. By Western blot analysis, endogenous beta-catenin levels were examined. By analyzing cytosolic fractions of PSEN1, transfected and nontransfected HEK 293 cells, and total brain extracts of AD patients and controls, we evaluated the effect of PSEN1 overexpression on beta-catenin stability. Finally, we analyzed the effect of pathogenic PSEN1 mutations on the interaction between PSEN1 and GSK3beta by co-immunoprecipitation experiments. RESULTS: We report reduced nuclear translocation of beta-catenin in cells stably expressing I143T, G384A, and T113-114ins PSEN1. The G384A PSEN1 mutation showed a similar pronounced effect on nuclear translocation of beta-catenin, as reported for processing of amyloid precursor protein (APP) into amyloid beta(Abeta). Overexpression of PSEN1 and the presence of pathogenic mutations in PSEN1 had no significant effect on the stability of beta-catenin. Nonspecific binding of overexpressed PSEN1 to endogenous GSK3beta was observed when GSK3beta was immunoprecipitated. Immunoprecipitation of PSEN1 in cells overexpressing PSEN1 and in native cells, however, did not result in co-immunoprecipitation of endogenous GSK3beta. CONCLUSION: Our results further establish the nuclear translocation assay of beta-catenin as an adequate alternative for traditional Abeta measurement to evaluate the effect of PSEN1 mutations on biochemical processes. We detected no significant effect of overexpressed wt or mt PSEN1 on the stability of beta-catenin. Finally, co-immunoprecipitation between PSEN1 and GSK3beta was not observed in our experimental setup.
The Wnt signaling pathway plays a major role in development, and upon deregulation it is implicated in neoplasia. The hallmark of the canonical Wnt signal is the protection of β-catenin from ubiquitination and proteasomal degradation induced by glycogen synthase kinase (GSK)-3β inhibition. The stabilized β-catenin translocates to the nucleus where it binds to T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors, activating the expression of Wnt target genes. In the absence of Wnt signal, TCF/LEF bind to Groucho (Gro)/TLE corepressors and repress Wnt target genes. Gro/TLE bind also to Engrailed (En) transcription factors mediating En-repressive activity on En target genes. Here, we present data suggesting that En-1 serves also as a negative regulator of β-catenin transcriptional activity; however, its repressive effect is independent of Gro/TLE. Our data suggest that En-1 acts by destabilizing β-catenin via a proteasomal degradation pathway that is GSK-3β–independent. Moreover, because En-1-mediated β-catenin degradation is also Siah independent, our data imply that En-1 exerts its repressive effect by a novel mechanism negatively controlling the level of β-catenin.
The Wnt/β-catenin signaling pathway plays essential roles in cell proliferation and differentiation, and deregulated β-catenin protein levels lead to many types of human cancers. On activation by Wnt, the Wnt co-receptor LDL receptor related protein 6 (LRP6) is phosphorylated at multiple conserved intracellular PPPSPXS motifs by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in recruitment of the scaffolding protein Axin to LRP6. As a result, β-catenin phosphorylation by GSK3 is inhibited and β-catenin protein is stabilized. However, how LRP6 phosphorylation and the ensuing LRP6-Axin interaction lead to the inhibition of β-catenin phosphorylation by GSK3 is not fully understood. In this study, we reconstituted Axin-dependent β-catenin phosphorylation by GSK3 and CK1 in vitro using recombinant proteins, and found that the phosphorylated PPPSPXS peptides directly inhibit β-catenin phosphorylation by GSK3 in a sequence and phosphorylation-dependent manner. This inhibitory effect of phosphorylated PPPSPXS motifs is direct and specific for GSK3 phosphorylation of β-catenin at Ser33/Ser37/Thr41 but not for CK1 phosphorylation of β-catenin at Ser45, and is independent of Axin function. We also show that a phosphorylated PPPSPXS peptide is able to activate Wnt/β-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of GSK3 in vivo. Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 places GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 phosphorylation of β-catenin. This model provides a possible mechanism to account, in part, for inhibition of β-catenin phosphorylation by Wnt-activated LRP6.
Glycogen synthase kinase-3 (GSK3) is a crucial enzyme contributing to the regulation of neuronal structure, plasticity and survival, is implicated as a contributory factor in prevalent diseases such as Alzheimer’s disease and mood disorders and is regulated by a wide range of signaling systems and pharmacological agents. Therefore, factors regulating GSK3 in vivo are currently of much interest. GSK3 is inhibited by phosphorylation of serine-9 or serine-21 in GSK3β and GSK3α, respectively. This study found that accurate measurements of phospho-Ser-GSK3 in brain are confounded by a rapid post-mortem dephosphorylation, with ~90% dephosphorylation of both GSK3 isoforms occurring within 2 min post-mortem. Furthermore, three anesthetics, pentobarbital, halothane and chloral hydrate, each caused large in vivo increases in the serine phosphorylation of both GSK3β and GSK3α in several regions of mouse brain. Thus, studies of the phosphorylation state of GSK3 in brain, and perhaps in other tissues, need to take into account post-mortem changes and the effects of anesthetics and there is a direct correlation between anesthesia and high levels of serine-phosphorylated GSK3.
anesthesia; glycogen synthase kinase-3; pentobarbital; post-mortem interval
The wnt pathway regulates the steady state level of β-catenin, a transcriptional coactivator for the Tcf3/Lef1 family of DNA binding proteins. We demonstrate that Tcf3 can inhibit β-catenin turnover via its competition with axin and adenomatous polyposis for β-catenin binding. A mutant of β-catenin that cannot bind Tcf3 is degraded faster than the wild-type protein in Xenopus embryos and extracts. A fragment of β-catenin and a peptide encoding the NH2 terminus of Tcf4 that block the interaction between β-catenin and Tcf3 stimulate β-catenin degradation, indicating this interaction normally plays an important role in regulating β-catenin turnover. Tcf3 is a substrate for both glycogen synthase kinase (GSK) 3 and casein kinase (CK) 1ε, and phosphorylation of Tcf3 by CKIε stimulates its binding to β-catenin, an effect reversed by GSK3. Tcf3 synergizes with CK1ε to inhibit β-catenin degradation, whereas CKI-7, an inhibitor of CK1ε, reduces the inhibitory effect of Tcf3. Finally, we provide evidence that CK1ε stimulates the binding of dishevelled (dsh) to GSk3 binding protein (GBP) in extracts. Along with evidence that a significant amount of Tcf protein is nonnuclear, these findings suggest that CK1ε can modulate wnt signaling in vivo by regulating both the β-catenin-Tcf3 and the GBP-dsh interfaces.
β-catenin; Tcf; wnt; casein kinase; Xenopus
Signaling by the Wnt family of secreted proteins plays an important role in animal development and is often misregulated in carcinogenesis. Wnt signal transduction is controlled by the rate of degradation of β-catenin by a complex of proteins including glycogen synthase kinase 3 (GSK3), adenomatous polyposis coli, and Axin. Dishevelled is required for Wnt signal transduction, and its activation results in stabilization of β-catenin. However, the biochemical events underlying this process remain largely unclear. Here we show that Xenopus Dishevelled (Xdsh) interacts with a Xenopus Axin-related protein (XARP). This interaction depends on the presence of the Dishevelled-Axin (DIX) domains in both XARP and Xdsh. Moreover, the same domains are essential for signal transduction through Xdsh. Finally, our data point to a possible mechanism for signal transduction, in which Xdsh prevents β-catenin degradation by displacing GSK3 from its complex with XARP.
Previous studies have indicated that Epstein-Barr virus (EBV) can modulate the Wnt pathway in virus-infected cells and this effect is mediated by EBV-encoded oncogene latent membrane protein 1 (LMP1). Here we have reassessed the role of LMP1 in regulating the expression of various mediators of the canonical Wnt cascade. Contradicting the previous finding, we found that the levels of E-cadherin, β-catenin, Glycogen Synthase Kinase 3ß (GSK3β), axin and α-catenin were not affected by the expression of LMP1 sequences from normal B cells or nasopharyngeal carcinoma. Moreover, we also show that LMP1 expression had no detectable effect on the E-cadherin and β-catenin interaction and did not induce transcriptional activation of β-catenin. Taken together these studies demonstrate that EBV-mediated activation of Wnt pathway is not dependent on the expression of LMP1.
In ischemic retinopathies, unrelieved hypoxia induces the formation of architecturally abnormal, leaky blood vessels that damage retina and ultimately can cause blindness. Because these newly formed blood vessels are functionally defective, they fail to alleviate underlying hypoxia, resulting in more pathological neovascularization and more damage to retina. With an established model of ischemic retinopathy, we investigated inhibition of glycogen synthase kinase-3β (GSK-3β) as a means for improving the architecture and functionality of pathological blood vessels in retina. In vitro, hypoxia increased GSK-3β activity in retinal endothelial cells, reduced β-catenin, and correspondingly impaired integrity of cell/cell junctions. Conversely, GSK-3β inhibitors restored β-catenin, improved cell/cell junctions, and enhanced the formation of capillary cords in three-dimensional collagen matrix. In vivo, GSK-3β inhibitors, at appropriately moderate doses, strongly reduced abnormal vascular tufts, reduced abnormal vascular leakage, and improved vascular coverage and perfusion during the proliferative phase of ischemia-driven retinal neovascularization. Most importantly, these improvements in neovasculature were accompanied by marked reduction in retinal hypoxia, relative to controls. Thus, GSK-3β inhibitors offer a promising strategy for alleviating retinal hypoxia by correcting key vascular defects typically associated with ischemia-driven neovascularization.
Retinopathy; Neovascularization; Glycogen synthase kinase-3β; Hypoxia; Endothelial cell; β-catenin