Regulation of glycogen synthase kinase 3beta and downstream Wnt signaling by axin.
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Axin is a recently identified protein encoded by the fused locus in mice that is required for normal vertebrate axis formation. We have defined a 25-amino-acid sequence in axin that comprises the glycogen synthase kinase 3beta (GSK-3beta) interaction domain (GID). In contrast to full-length axin, which has been shown to antagonize Wnt signaling, the GID inhibits GSK-3beta in vivo and activates Wnt signaling. Similarly, mutants of axin lacking key regulatory domains such as the RGS domain, which is required for interaction with the adenomatous polyposis coli protein, bind and inhibit GSK-3beta in vivo, suggesting that these domains are critical for proper regulation of GSK-3beta activity. We have identified a novel self-interaction domain in axin and have shown that formation of an axin regulatory complex in vivo is critical for axis formation and GSK-3beta activity. Based on these data, we propose that the axin complex may directly regulate GSK-3beta enzymatic activity in vivo. These observations also demonstrate that alternative inhibitors of GSK-3beta can mimic the effect of lithium in developing Xenopus embryos. (+info)
Membrane-anchored plakoglobins have multiple mechanisms of action in Wnt signaling.
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In Wnt signaling, beta-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing gene Siamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic beta-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize beta-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of beta-catenin turnover. Expression of cnxPg increases levels of cytosolic beta-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize beta-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with beta-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both beta-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm of Xenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on beta-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity. (+info)
Suppression of glycogen synthase kinase activity is not sufficient for leukemia enhancer factor-1 activation.
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Glycogen synthase kinase-3 (GSK) can be regulated by different signaling pathways including those mediated by protein kinase Akt and Wnt proteins. Wnt proteins are believed to activate a transcription factor leukemia enhancer factor-1 (LEF-1) by inhibiting GSK, and Akt was shown to phosphorylate GSK and inhibit its kinase activity. We investigated the effect of an activated Akt on the accumulation of cytosolic beta-catenin and LEF-1-dependent transcription. Although the activated Akt, mAkt, clearly inhibited the kinase activity of GSK, mAkt alone did not induce accumulation of cytosolic beta-catenin or activate LEF-1-dependent transcription. On the contrary, coexpressed Wnt-1 and Frat activated LEF-1 but did not show significant inhibition of GSK-mediated phosphorylation of a peptide substrate. However, mAkt could act synergistically with Wnt-1 or Frat to activate LEF-1. In addition, the interaction of GSK for Axin appeared to decrease in the presence of mAkt, whereas the interaction for Frat remained unchanged. Consistently, a GSK mutant with substitution of a Phe residue for residue Tyr-216, which showed one-fifth of kinase activity of the wild-type GSK, exhibited a reduced association for Axin than the wild-type GSK. These results suggest that inhibition of GSK kinase activity is not sufficient for activation of LEF-1 but may facilitate the activation by reducing the interaction of GSK for Axin. The additional mechanism for LEF-1 activation may require dissociation of GSK from Axin as Frat facilitates the dissociation of GSK from Axin. (+info)
Relationship of vegetal cortical dorsal factors in the Xenopus egg with the Wnt/beta-catenin signaling pathway.
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In Xenopus, the dorsal factor in the vegetal cortical cytoplasm (VCC) of the egg is responsible for axis formation of the embryo. Previous studies have shown that VCC dorsal factor has properties similar to activators of the Wnt/beta-catenin-signaling pathway. In this study, we examined the relationship of the VCC dorsal factor with components of the pathway. First, we tested whether beta-catenin protein, which is known to be localized on the dorsal side of early embryos, accounts for the dorsal axis activity of VCC. Reduction of beta-catenin mRNA and protein in oocytes did not diminish the activity of VCC to induce a secondary axis in recipient embryos. The amount of beta-catenin protein was not enriched in VCC compared to animal cortical cytoplasm, which has no dorsal axis activity. These results indicate that beta-catenin is unlikely to be the VCC dorsal axis factor. Secondly, we examined the effects of four Wnt-pathway-interfering constructs (dominant-negative Xdsh, XGSK3, Axin, and dominant-negative XTcf3) on the ability of VCC to induce expression of the early Wnt target genes, Siamois and Xnr3. The activity of VCC was inhibited by Axin and dominant negative XTcf3 but not by dominant negative Xdsh or XGSK3. We also showed that VCC decreased neither the amount nor the activity of exogenous XGSK3, suggesting that the VCC dorsal factor does not act by affecting XGSK3 directly. Finally, we tested six Wnt-pathway activating constructs (Xwnt8, Xdsh, dominant negative XGSK3, dominant negative Axin, XAPC and beta-catenin) for their responses to the four Wnt-pathway-interfering constructs. We found that only XAPC exhibited the same responses as VCC; it was inhibited by Axin and dominant negative XTcf3 but not by dominant negative Xdsh or XGSK3. Although the connection between XAPC and the VCC dorsal factor is not yet clear, the fact that APC binds Axin suggests that the VCC dorsal factor could act on Axin rather than XGSK3. (+info)
Axin forms a complex with MEKK1 and activates c-Jun NH(2)-terminal kinase/stress-activated protein kinase through domains distinct from Wnt signaling.
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Axin negatively regulates the Wnt pathway during axis formation and plays a central role in cell growth control and tumorigenesis. We found that Axin also serves as a scaffold protein for mitogen-activated protein kinase activation and further determined the structural requirement for this activation. Overexpression of Axin in 293T cells leads to differential activation of mitogen-activated protein kinases, with robust induction for c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase, moderate induction for p38, and negligible induction for extracellular signal-regulated kinase. Axin forms a complex with MEKK1 through a novel domain that we term MEKK1-interacting domain. MKK4 and MKK7, which act downstream of MEKK1, are also involved in Axin-mediated JNK activation. Domains essential in Wnt signaling, i. e. binding sites for adenomatous polyposis coli, glycogen synthase kinase-3beta, and beta-catenin, are not required for JNK activation, suggesting distinct domain utilization between the Wnt pathway and JNK signal transduction. Dimerization/oligomerization of Axin through its C terminus is required for JNK activation, although MEKK1 is capable of binding C terminus-deleted monomeric Axin. Furthermore, Axin without the MEKK1-interacting domain has a dominant-negative effect on JNK activation by wild-type Axin. Our results suggest that Axin, in addition to its function in the Wnt pathway, may play a dual role in cells through its activation of JNK/stress-activated protein kinase signaling cascade. (+info)
Protein phosphatase 2Calpha dephosphorylates axin and activates LEF-1-dependent transcription.
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The Dishevelled (Dvl) gene family encodes cytoplasmic proteins that are necessary for Wnt signal transduction. Utilizing the yeast two-hybrid system, we identified protein phosphatase 2Calpha (PP2C) as a Dvl-PDZ domain-interacting protein. PP2C exists in a complex with Dvl, beta-catenin, and Axin, a negative regulator of Wnt signaling. In a Wnt-responsive LEF-1 reporter gene assay, expression of PP2C activates transcription and also elicits a synergistic response with beta-catenin and Wnt-1. In addition, PP2C expression relieves Axin-mediated repression of LEF-1-dependent transcription. PP2C utilizes Axin as a substrate both in vitro and in vivo and decreases its half-life. These results indicate that PP2C is a positive regulator of Wnt signal transduction and mediates its effects through the dephosphorylation of Axin. (+info)
Biochemical interactions in the wnt pathway.
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The wnt signal transduction pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. The cytoplasmic component beta-catenin is central to the transmission of wnt signals to the nucleus: in the absence of wnts beta-catenin is constitutively degraded in proteasomes, whereas in the presence of wnts beta-catenin is stabilized and associates with HMG box transcription factors of the LEF/TCF family. In tumors, beta-catenin degradation is blocked by mutations of the tumor suppressor gene APC (adenomatous polyposis coli), or of beta-catenin itself. As a consequence, constitutive TCF/beta-catenin complexes are formed and activate oncogenic target genes. This review discusses the mechanisms that silence the pathway in cells that do not receive a wnt signal and goes on to describe the regulatory steps involved in the activation of the pathway. (+info)
Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification.
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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. (+info)