The activity of APC was assayed in the early Xenopus embryo, which has been established as a good model for the analysis of the signaling activity of the APC-associated protein beta-catenin. When expressed in the future ventral side of a four-cell embryo, full-length APC induces a secondary dorsoanterior axis and the induction of the homeobox gene Siamois. This is similar to the phenotype previously observed for ectopic beta-catenin expression. In fact, axis induction by APC requires the availability of cytosolic beta-catenin. These results indicate that APC has signaling activity in the early Xenopus embryo. Signaling activity resides in the central domain of the protein, a part of the molecule that is missing in most of the truncating APC mutations in colon cancer. Signaling by APC in Xenopus embryos is not accompanied by detectable changes in expression levels of beta-catenin, indicating that it has direct positive signaling activity, in addition to its role in beta-catenin turnover. From these results a model is proposed in which APC acts as part of the Wnt/beta-catenin signaling pathway, either upstream of, or in conjunction with, beta-catenin (Vleminckx, 1997).

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, the relationship of components of the pathway to the VCC dorsal factor was examined. Initially examined was 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 does not diminish the activity of VCC to induce a secondary axis in recipient embryos. The amount of beta-catenin protein is 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. The next series of studies looked at 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 is inhibited by Axin and dominant negative XTcf3 but not by dominant negative Xdsh or XGSK3. VCC decreases neither the amount nor the activity of exogenous XGSK3, suggesting that the VCC dorsal factor does not act by affecting XGSK3 directly. Finally, six Wnt-pathway activating constructs (Xwnt8, Xdsh, dominant negative XGSK3, dominant negative Axin, XAPC and beta-catenin) were tested for their responses to the four Wnt-pathway-interfering constructs. Only XAPC exhibits the same responses as VCC; it is 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 might act on Axin rather than XGSK3 (Marikawa, 1999).

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, GSK-3 binding protein (GBP or FRAT1 ), which is required for axis formation in Xenopus, binds and inhibits GSK-3. It has been shown that GBP inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, a dominant-negative GSK-3 mu- tant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating GSK-3's catalytic activity. 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 a 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 (Farr, 2000).

The coordinated regulation of GSK-3 activity by both positive and negative regulators is critical for a wide range of downstream effects, from insulin regulation to developmental processes. The experiments presented here advance the understanding of GSK-3 regulation and suggest the following model for Xgsk-3 regulation in the fertilized Xenopus egg. On the ventral side, beta-catenin levels are kept low by Xgsk-3-dependent phosphorylation in a complex including Axin and APC. Phosphorylation of Axin and APC by Xgsk-3 may be required to assemble this complex. On the dorsal side, GBP inhibits phosphorylation of beta-catenin either by displacing Xgsk-3 from the Axin/APC/Xgsk-3 complex or by prebinding GSK-3 and preventing its association with the Axin-APC complex. GBP binding to Xgsk-3 may block access of protein substrates to the active site. GBP might also prevent Xgsk-3 from phosphorylating Axin and APC, and thereby further inhibit complex formation. The dominant-negative Xgsk-3 acts in a manner analogous to GBP since it either displaces wild-type Xgsk-3 from the complex, or binds endogenous Xgsk-3 and prevents its binding to Axin, thus, inhibiting beta-catenin degradation. The complete chain of molecular events linking fertilization and the dorsal enrichment of beta-catenin has yet to be determined, but this study brings closer an understanding the interactions of key players. It will ultimately be very interesting to elucidate the relative dorso-ventral abundance of each of these proteins in the Xenopus oocyte and fertilized embryo. However, it may not be just the relative abundance of each of these players that specifies the endogenous axis. Alternatively, posttranslational modifications and/or the presence of yet unidentified factors could affect dorso-ventral differences in known players. GBP is a candidate for the dorsal determinant, and the reagents to examine the possible dorsal localization or dorsal modification of GBP are being developed. Alternatively, GBP may be ubiquitous, but be activated or recruited to function by another factor that becomes dorsally localized in response to sperm entry. The determinant might, for example, bind to the Axin/APC/Xgsk-3 complex and alter its conformation in such a way as to allow GBP to bind Xgsk-3 and remove it from the complex. In this regard, the observation that Dishevelled interacts with GBP and Axin and is dorsally enriched in the early Xenopus embryo is very suggestive of such a mechanism. Alternatively, GBP might function together with Dishevelled to prevent the initial formation of complexes between Axin and GSK-3 in the dorsal cortical cytoplasm, rather than functioning to disrupt complexes once they have formed (Farr, 2000).

Regulation of beta-catenin degradation by intracellular components of the wnt pathway was reconstituted in cytoplasmic extracts of Xenopus eggs and embryos. The ubiquitin-dependent beta-catenin degradation in extracts displays a biochemical requirement for axin, GSK3, and APC. Axin dramatically accelerates while dishevelled inhibits beta-catenin turnover. Through another domain, dishevelled recruits GBP/Frat1 to the APC-axin-GSK3 complex. These results confirm and extend models in which inhibition of GSK3 has two synergistic effects: (1) reduction of APC phosphorylation and loss of affinity for beta-catenin and (2) reduction of beta-catenin phosphorylation and consequent loss of its affinity for the SCF ubiquitin ligase complex. Dishevelled thus stabilizes beta-catenin, which can dissociate from the APC/axin complex and participate in transcriptional activation (Salic, 2000).

The central mechanistic features of beta-catenin phosphorylation and ubiquitination have been well described in several systems by genetic approaches and by overexpression in cell lines and embryos. These studies have identified dsh, axin, GSK3, APC, and GBP/FRAT1 as important regulators of beta-catenin levels. However, there is disagreement as to whether APC acts in a positive or negative fashion; whether GBP plays an important role, or whether it just buffers GSK3; how the components interact; what the precise function of axin and APC is in regulating beta-catenin phosphorylation, and finally, the role of dsh in this process. To address these issues, cytoplasmic extracts from Xenopus eggs were used to reconstitute the regulated degradation of beta-catenin. By measuring beta-catenin stability directly rather than steady-state levels of the protein, the complicating effects of transcription and translation present in other systems were avoided. By adding known amounts of purified proteins to extracts and measuring degradation kinetics, a quantitative analysis of wnt signaling can be performed. This system also allows an examination of mutant proteins that might not show phenotypes if expressed in vivo, due to rapid turnover. In extracts, beta-catenin was degraded with a half-life of 1 hr, dropping to less than 15 min in the presence of added axin, which suggestes that axin is rate limiting for degradation. Addition of dsh, a positive regulator epistatically upstream of GSK3, stabilizes beta-catenin, suggesting that the wnt pathway from dsh to beta-catenin had been recapitulated in extracts (Salic, 2000).

This in vitro system allowed for a test of the biochemical requirement for several different components of the wnt pathway, ruling out the necessary involvement of any machinery upstream of protein translation. beta-catenin degradation depends on axin, GSK3, and APC; extracts depleted of any of these proteins cannot degrade beta-catenin. Of the four defined domains in axin, only the C-terminal DIX domain is dispensable for activity. The DIX domain of axin binds a related domain in dsh, and this interaction is required for dsh function; hence, binding of axin and dsh through the DIX domains confers signal-dependent regulation of beta-catenin. Another domain of dsh required for activity is PDZ, which binds GBP. Dsh and GBP synergize to inhibit beta-catenin degradation in extracts and beta-catenin phosphorylation in vitro, which are both axin-dependent processes. The inhibitory effect of GBP and dsh on beta-catenin degradation in extracts is reversed by high concentrations of GSK3 due to titration of dsh/GBP on axin. These observations further support a role for dsh, as an adaptor protein recruiting GBP to the axin/GSK3/APC/beta-catenin complex and inhibiting locally the enzymatic activity of GSK3. These recent results suggest that the axin-GSK3 interaction is highly dynamic and that increasing the local GBP concentration at the level of the complex efficiently promotes the dissociation of GSK3 from its site on axin (Salic, 2000).

The simplest model would have beta-catenin and GSK3 binding to their sites on axin and GBP binding to its site on dsh. When dsh and axin bind via their DIX domains, GSK3 would be inhibited. How does APC fit into this model? In addition to the mechanistic confusions about the role of APC, there are suggestions that APC can be an inhibitor of beta-catenin degradation in some circumstances or an activator in others. Specifically in Xenopus, injection of APC can cause secondary axis formation, the opposite of the ventralizing effects of axin injections. In an in vitro system, there is an absolute requirement for APC in beta-catenin degradation, consistent with genetic experiments in Drosophila and experiments in cultured cells. The confusion is likely to be simply due to the tendency of overexpressed APC to act as a dominant inhibitor. In Xenopus extracts, overexpressed APC is a potent inhibitor of beta-catenin degradation, although these experiments yield a mixture of truncated and full-length APC proteins. The role of APC in beta-catenin degradation is clear from the in vitro experiments. An interaction of APC with the RGS domain of axin is essential for beta-catenin degradation, demonstrating that endogenous APC is in fact a negative regulator of beta-catenin levels in Xenopus. Although beta-catenin binds to axin beads in a purified system, the two proteins show little interaction in the nanomolar range. Extracts contain an activity that promotes the binding of beta-catenin to axin at low concentrations. The activity is inhibited by the RGS domain of axin, suggesting that it is either APC or APC complexed to other proteins. Also, axinDeltaRGS (which cannot bind APC) does not bind beta-catenin in extracts. With purified components in vitro, APC accelerates the binding of beta-catenin to axin, recapitulating the stimulating effect of extracts on binding between the two proteins. Taken together, these results identify APC as the activity stimulating the axin-beta-catenin interaction in extracts (Salic, 2000).

beta-catenin interacts with numerous cellular proteins, some not directly involved in wnt signaling. The requirement for an APC-like molecule makes sense if one realizes that not only the stability of beta-catenin is important but also its availability for other interactions, both transcriptional and cytoskeletal. Aside from participating in beta-catenin degradation, APC also prevents the interaction of beta-catenin with Tcf3, thus inhibiting the transcriptional activation function of beta-catenin. Additionally, APC maintains a pool of beta-catenin in a state that can either lead to its degradation or release in response to a wnt signal. The phosphorylation state of APC is maintained by rapid futile cycles of opposing phosphorylation and dephosphorylation reactions. Changes in the phosphorylation of APC by GSK3 acts as a switch to trigger either beta-catenin degradation or its discharge in the cytoplasm. Although it has been suggested that phosphorylation of axin modulates binding to beta-catenin, no evidence could be found for such a regulation in extracts because (1) APC is the principal mediator of the axin-beta-catenin interaction and (2) using purified axin and beta-catenin, an increase in the affinity of beta-catenin for axin due to phosphorylation of the latter by GSK3 could not be detected. These data suggests that the APC/axin/GSK3/beta-catenin complex exists in two states. In one state, beta-catenin is bound tightly to axin via APC; beta-catenin and APC are both phosphorylated by GSK3. In this situation, phosphorylated beta-catenin can only be removed through SCF-dependent degradation while phosphorylated APC continues to bind beta-catenin molecules avidly. Upon binding of active dsh to axin, GBP interacts with GSK3, removing it from axin competitively. This inhibits phosphorylation on both beta-catenin and APC. The former stabilizes beta-catenin to ubiquitination, and the latter releases intact beta-catenin so that it can interact with other partners. The beta-catenin-binding site on axin is also important because its deletion impairs the function of axin. Although this scenario is written as a set of irreversible steps, it is likely that all binding events are dynamic. The fact that dominant-negative GSK3 blocks degradation of beta-catenin in extracts suggests that the interaction between endogenous axin and GSK3 must be dynamic. The dissociation rate for GSK3 bound to axin must be fast enough to allow its displacement by the dominant-negative mutant on a time scale of perhaps less than 1 hr. The same must be true for the axin-APC interaction, since the RGS domain of axin (which interacts with APC) blocks beta-catenin degradation (Salic, 2000).

The stability of beta-catenin is subject to a system of weak protein interactions and posttranslational modifications. The ability to dissect this pathway in vitro without altering appreciably its kinetics and without simplifying essential steps should aid an understanding of these weak interactions and their physiological consequences. Several remaining questions should be clarified by studies in partially purified systems and ultimately by reconstitution from purified components. These include the mechanism of signal transmission from frizzled to dsh and the mechanism of dsh activation. In particular, does the wnt signal regulate the GBP-dsh or the dsh-axin interaction? It will also be of interest to know whether APC suffices by itself to mediate beta-catenin degradation or whether associated proteins are also required. Finally, it will be of interest to know whether signaling pathways cross-regulate the wnt pathway and how that might work. In vitro reconstitution should be useful in deciphering the molecular events involved in transducing the wnt signal (Salic, 2000).

During the first few cleavages of the Caenorhabditis elegans embryo, localized expression of factors that regulate transcription or that mediate cell-cell interactions results in each blastomere acquiring a distinct identity, or potential to differentiate. Each blastomere then executes a unique and nearly invariant lineage, producing numerous cell types through a series of predominantly anterior/posterior (a/p) cleavages. Because blastomere lineages are essentially invariant, this means that patterns of cell division are correlated reproducibly with specific patterns of cell differentiation. For example, in the lineage of a blastomere called MS, the MS descendant born from the division sequence p-a-a-p-p invariably undergoes programmed cell death or apoptosis; none of the other MS descendants born at the same time, but from different division sequences, undergo apoptosis. Within a lineage, how is cell type differentiation reproducibly matched with division sequence? Invariant cleavage patterns could place cells consistently in the same position with respect to determinative environmental signals in the embryo. However, several studies have shown that, after about the 12-cell stage of embryogenesis, blastomeres have remarkable abilities to execute their normal lineages even after neighboring blastomeres are killed or removed. For example, the MS descendant born from the division sequence p-a-a-p-p undergoes apoptosis even if every blastomere except for MS is killed. Thus, in some lineages, cell fates do not appear to be determined by external, environmental cues within the embryo (Lin, 1998).

The pop-1 gene, coding a Tcf-1 and Lef-1 related protein, is part of a general system for transducing information about division sequences into changes in the cell nucleus that affect differentiation. The pop-1 gene was identified originally because of its role in the development of the MS blastomere. MS normally produces mesodermal tissues, and its sister E produces only endoderm. In a pop-1 mutant, MS adopts an E-like fate and produces endoderm. A signaling pathway similar to the Wnt pathway of vertebrates and Drosophila melanogaster has been shown to be required for MS and E to have different fates. In models for Wnt signaling, reception of the Wnt signal results in the nuclear localization of a beta-catenin such as the Armadillo protein in Drosophila; a C. elegans homolog WRM-1 is required for MS and E differences, but the localization of WRM-1 has not yet been determined. Once in the nucleus, beta-catenin is thought to interact with constitutive nuclear proteins such as Tcf-1 in vertebrates or the related Pangolin in Drosophila to regulate transcription; the POP-1 protein in C. elegans has sequence similarity to Tcf-1 and acts downstream in the Wnt-like pathway. A polyclonal antiserum raised against the POP-1 protein shows a slightly lower level of staining in the E nucleus than in the MS nucleus in most, but not all, C. elegans embryos. A monoclonal antibody specific to the POP-1 protein shows different levels of nuclear staining in almost all a/p pairs of sister blastomeres in the early embryo, including the MS/E pair. In each of these a/p pairs, a higher level of POP-1 staining is detected in the anterior sister than in the posterior sister. Loss of pop-1(+) activity causes several anterior cells to adopt fates similar to the fates of their posterior sisters. These studies show that the Wnt-like signaling pathway is required for generating or interpreting a/p polarity throughout the early C. elegans embryo and that POP-1 appears to be part of a general mechanism that couples division sequence to different patterns of gene expression in sister cells born from a/p cleavages (Lin, 1998).

The MS/E decision requires components of a Wnt-like signaling pathway. Studies in vertebrates and Drosophila have led to a model in which Wnt signaling regulates an interaction between beta-catenin and POP-1-related proteins, such as Tcf-1 or Lef-1. Additional studies indicate that the APC (human adenomatous polyposis) protein also can regulate beta-catenin, but it has not been resolved whether APC acts downstream, or in parallel to, Wnt. In C. elegans, the loss of wild-type activity of the wrm-1 (beta-catenin) gene alone, or the simultaneous loss of mom-2 (Wnt) and apr-1 (APC) activities, prevents the MS/E decision and causes MS and E to have similar levels of POP-1. Therefore, it was asked whether these genes are required for POP-1 asymmetry in other a/p pairs of sisters. All a/p pairs of sister blastomeres appear to have equivalent levels of POP-1 in wrm-1(RNAi) embryos (RNAi refers to RNA interference, which produces mutant-like phenotypes on treated embryos) and in mom-2(or9);apr-1(RNAi) embryos. Surprisingly, mom-2(or9) single mutants retain POP-1 asymmetry in AB descendants, though they lacked POP-1 asymmetry in the MS/E blastomeres. Thus, WRM-1 is essential for all POP-1 asymmetry (Lin, 1998).

A novel member of the human frizzled (Fz) gene family was cloned and found to be specifically expressed in 3 of 13 well differentiated (23%), 13 of 20 moderately differentiated (62%), and 12 of 14 poorly differentiated (86%) squamous cell esophageal carcinomas compared with the adjacent uninvolved normal mucosa. The FzE3 cDNA encodes a protein of 574 amino acids and shares high sequence homology with the human FzD2 gene, particularly in the putative ligand binding region of the cysteine-rich extracellular domain. Functional analysis reveals that transfection and expression of the FzE3 cDNA in esophageal carcinoma cells stimulates complex formation between adenomatous polyposis coli (APC) and beta-catenin followed by nuclear translocation of beta-catenin. Furthermore, cotransfection of a mutant construct encoding a FzE3 protein with a C-terminal truncation completely inhibits the interaction of APC with beta-catenin in cells. Finally, coexpression of FzE3 with Lef-1 transcription factor enhances beta-catenin translocation to the nucleus. These observations suggest that FzE3 gene expression may down-regulate APC function and enhance beta-catenin mediated signals in poorly differentiated human esophageal carcinomas (Tanaka, 1998).

Dysregulation of Wnt-beta-catenin signaling disrupts axis formation in vertebrate embryos and underlies multiple human malignancies. The adenomatous polyposis coli (APC) protein, axin, and glycogen synthase kinase 3beta form a Wnt-regulated signaling complex that mediates the phosphorylation-dependent degradation of beta-catenin. A protein phosphatase 2A (PP2A) regulatory subunit, B56, interacts with APC in the yeast two-hybrid system. Expression of B56 reduces the abundance of beta-catenin and inhibits transcription of beta-catenin target genes in mammalian cells and Xenopus embryo explants. The B56-dependent decrease in beta-catenin is blocked by oncogenic mutations in beta-catenin or APC, and by proteasome inhibitors. B56 may direct PP2A to dephosphorylate specific components of the APC-dependent signaling complex and thereby inhibit Wnt signaling. Loss of PP2A function may provide an additional route to activation of Wnt signaling and oncogenesis. Consistent with this, mutations in the gene encoding the beta isoform of the PP2A subunit have been identified in colon and lung cancers (Seeling, 1999).

The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. This study shows that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, the functional importance of FAK was investigated following deletion of the Apc tumor suppressor protein within the intestinal epithelium. Following Apc loss, FAK expression increased in a c-Myc-dependent manner. Codeletion of Apc and Fak strongly reduced proliferation normally induced following Apc loss, and this was associated with reduced levels of phospho-Akt and suppression of intestinal tumorigenesis in Apc heterozygous mice. Thus, FAK is required downstream of Wnt Signaling, for Akt/mTOR activation, intestinal regeneration, and tumorigenesis. Importantly, this work suggests that FAK inhibitors may suppress tumorigenesis in patients at high risk of developing colorectal cancer (Ashton, 2010).