APC cloning and expression

Mammalian APC2, which closely resembles APC in overall domain structure, has been cloned and functionally analyzed and shown to contain two SAMP domains, both of which are required for binding to conductin. Like APC, APC2 regulates the formation of active betacatenin-Tcf complexes, as demonstrated using transient transcriptional activation assays in APC -/- colon carcinoma cells. Human APC2 maps to chromosome 19p13.3. APC and APC2 may therefore have comparable functions in development and cancer (van Es, 1999).

The APC gene is mutated in familial adenomatous polyposis and in sporadic colorectal tumours. The APC gene product is a 300,000 mol. wt cytoplasmic protein that binds to at least three other proteins: beta-catenin, a cytoplasmic E-cadherin-associated protein; hDLG, a human homolog of the Drosophila Discs large tumour suppressor protein, and glycogen synthase kinase 3 beta, a mammalian homolog of the Drosophila Shaggy/Zeste white 3 protein. The adenomatous polyposis coli gene is highly expressed in the brain, suggesting that it may be involved in nerve function. Apc is localized in the pericapillary astrocytic endfeet throughout the mouse central nervous system. Apc is also localized in the astrocytic processes in the cerebellar granular layer, and displays concentrated expression in the terminal plexuses of the basket cell fibers around Purkinje cells. Apc is further expressed in neuronal cell bodies and/or nerve fibres in the olfactory bulb, hippocampus, brain stem, spinal cord and dorsal root ganglia. Apc is co-localized with beta-catenin and/or hDLG in neurons and nerve fibers, but not in astrocytes. From these results, Apc is suggested to participate in a signal transduction pathway in astrocytes that is independent of beta-catenin and hDLG, as well as in the regulation of neuronal functions in association with beta-catenin and hDLG (Senda, 1998).

APC mutation

According to the classical interpretation of Knudson's 'two-hit' hypothesis for tumorigenesis, the two 'hits' are independent mutation events, the end result of which is loss of a tumor suppressing function. Recently, it has been shown that the APC (adenomatous polyposis coli) gene does not entirely follow this model. Both the position and type of the second hit in familial adenomatous polyposis (FAP) polyps depend on the localization of the germline mutation. This non-random distribution of somatic hits has been interpreted as the result of selection for more advantageous mutations during tumor formation. However, APC encodes a multifunctional protein, and the exact cellular function upon which this selection is based is yet unknown. In this study, somatic APC point mutations and loss of heterozygosity (LOH) were examined in 133 colorectal adenomas from six FAP patients. It was observed that when germline mutations result in truncated proteins without any of the seven ß-catenin downregulating 20-amino-acid repeats distributed in the central domain of APC, the majority of the corresponding somatic point mutations retain one or, less frequently, two of the same 20-amino-acid repeats. Conversely, when the germline mutation results in a truncated protein retaining one 20-amino-acid repeat, most second hits remove all 20-amino-acid repeats. The latter is frequently accomplished by allelic loss. Notably, and in contrast to previous observations, in a patient where the germline APC mutation retains two such repeats, the majority of the somatic hits are point mutations (and not LOH) located upstream and removing all of the 20-amino-acid repeats. These results indicate selection for APC genotypes that are likely to retain some activity in downregulating ß-catenin signaling. It is proposed that this selection process is aimed at a specific degree of ß-catenin signaling optimal for tumor formation, rather than at its constitutive activation by deletion of all of the ß-catenin downregulating motifs in APC (Albuquerque, 2002).

Although Apc is well characterized as a tumor-suppressor gene in the intestine, the precise mechanism of this suppression remains to be defined. Using a novel inducible Ahcre transgenic line in conjunction with a loxP-flanked Apc allele, loss of Apc is shown to acutely activate Wnt signaling through the nuclear accumulation of ß-catenin. Coincidentally, it perturbs differentiation, migration, proliferation, and apoptosis, such that Apc-deficient cells maintain a 'crypt progenitor-like' phenotype. Critically, a series of Wnt target molecules has been confirmed in an in vivo setting, and a series of new candidate targets has been identified within the same setting (Sansom, 2004).

ß-catenin levels were examined within the Cre+Apcfl/fl tissue at day 5. There was no increase in total ß-catenin in the Cre+Apcfl/fl samples. However, levels of dephosphorylated ß-catenin were moderately elevated and, crucially, ß-catenin relocalized to the nuclei in the Cre+Apcfl/fl tissue. To more precisely define the time scale of nuclear relocalization, immunohistochemical analyses were performed at days 1, 2, 3, and 4 following induction of the cre recombinase. This analysis showed that relocalization occurred at day 3, and this was coincident with the observed onset of changes in morphology, proliferation, and apoptosis (Sansom, 2004).

To test whether nuclear ß-catenin was activating transcription of its known target genes, microarray analysis was performed using the affymetrix U74A chip. RNA samples were derived from sibling Cre+Apcfl/fl and Cre+Apc+/+ mice given four daily injections of ß-napthoflavone and killed at days 4 and 5. Of the 100 most significantly up-regulated genes, 10 have been associated with Wnt signaling (either directly or through arrays that had examined targets of the ß-catenin/TCF4 complex). Of the comparable genes up-regulated at day 5, 45 of 47 showed increases, of which 36 were in excess of twofold. These 36 included c-Myc, CD44, Tiam 1, Sema3c, and EphB3, all of which were confirmed changes at day 5. These data are, therefore, consistent with the notion that these are important early changes following nuclear relocalization of ß-catenin. Use of the larger chip set also revealed up-regulation of other Wnt target genes at day 4, including Sox17 and Axin2 (Sansom, 2004).

To validate the results obtained from the microarray analysis, the expression pattern of a subset of dysregulated genes was examined. Up-regulation of CD44, C-Myc, laminin gamma2, EphB2, and EphB3 was confirmed immunohistochemically in the Apc-deficient tissue. Expression of the EphrinB2 ligand, which is normally restricted to the top of the crypts and villi, was reduced in concordance with the reduction in villus differentiation in the Cre+Apcfl/fl tissue. These data therefore confirm, in an in vivo setting, many of the targets of Wnt signaling that have been implicated from in vitro studies. These include up-regulation of CD44, c-Myc, MMP-7 (matrilysin), gamma-2 laminin, Sema3c (confirmed by RT-PCR) Ets-2, EphB2, EphB3, and GPR49. The array analysis also indicates up-regulation of a series of genes that either interact with CD44 or are targets of CD44. These include MMP-7, TIAM1, FGF4 and its receptor, and TASR-2. The up-regulation of TIAM1 is particularly interesting, since TIAM1 has been shown to mediate Ras signaling. Indeed, mice deficient in TIAM1 are resistant to Ras-induced skin tumors (Sansom, 2004).

Deficiency of EphB3 has been shown to lead to abnormal Paneth cell positioning in the crypt. Wnt-mediated up-regulation of EphB3 yields a similar Paneth cell phenotype, confirming a pivotal role for the EphB/ephrinB mutual repulsion system in defining crypt-villus architecture. These results are also consistent with the notion that Apc mutant cells express the same genetic program as cells at positions 1-2 of the crypt, with notable increases in EphB3, MMP7, and Pla2g2a being characteristic of both Paneth cells and the Apc-deficient cells described in this study (Sansom, 2004).

In summary, Apc has been shown to be a critical determinant of cell fate in the murine small intestinal epithelium. Acute activation of Wnt signaling immediately produces many of the phenotypes associated with early colorectal lesions: failed differentiation, increased proliferation, and aberrant migration. Within a short time scale, multiple processes are affected: interactions with the cellular matrix, interactions with the basement membrane, increased proliferation, and failure of positional cues (EphB/ephrinB) (Sansom, 2004).

Loss of Apc appears to be one of the major events initiating colorectal cancer. However, the first events responsible for this initiation process are not well defined and the ways in which different epithelial cell types respond to Apc loss are unknown. A conditional gene-ablation approach in transgenic mice expressing tamoxifen-dependent Cre recombinase all along the crypt-villus axis was used to analyze the immediate effects of Apc loss in the small intestinal epithelium, both in the stem-cell compartment and in postmitotic epithelial cells. Within 4 days, Apc loss induced a dramatic enlargement of the crypt compartment associated with intense cell proliferation, apoptosis and impairment of cell migration. This result confirms the gatekeeper role of Apc in the intestinal epithelium in vivo. Although Apc deletion activated ß-catenin signaling in the villi, neither proliferation nor morphological change was observed in this compartment. This highlights the dramatic difference in the responses of immature and differentiated epithelial cells to aberrant ß-catenin signaling. These distinct biological responses were confirmed by molecular analyses, revealing that Myc and cyclin D1, two canonical ß-catenin target genes, were induced in distinct compartments. Apc is a crucial determinant of cell fate in the murine intestinal epithelium. Apc loss perturbs differentiation along the enterocyte, goblet and enteroendocrine lineages, and promotes commitment to the Paneth cell lineage through ß-catenin/Tcf4-mediated transcriptional control of specific markers of Paneth cells, the cryptdin/defensin genes (Andreu, 2005).

The tumor suppressor gene Apc is a member of the Wnt signaling pathway that is involved in development and tumorigenesis. Heterozygous knockout mice for Apc have a tumor predisposition phenotype and homozygosity leads to embryonic lethality. To understand the role of Apc in development a floxed allele was generated. These mice were mated with a strain carrying Cre recombinase under the control of the human Keratin 14 (K14) promoter, which is active in basal cells of epidermis and other stratified epithelia. Mice homozygous for the floxed allele that also carry the K14-cre transgene were viable but had stunted growth and died before weaning. Histological and immunochemical examinations revealed that K14-cre-mediated Apc loss resulted in aberrant growth in many ectodermally derived squamous epithelia, including hair follicles, teeth, and oral and corneal epithelia. In addition, squamous metaplasia was observed in various epithelial-derived tissues, including the thymus. The aberrant growth of hair follicles and other appendages as well as the thymic abnormalities in K14-cre; Apc(CKO/CKO) mice suggest the Apc gene is crucial in embryonic cells to specify epithelial cell fates in organs that require epithelial-mesenchymal interactions for their development (Kuraguchi, 2006).

In the postimplantation mouse embryo, axial patterning begins with the restriction of expression of a set of genes to the distal visceral endoderm (DVE). This proximodistal (PD) axis is subsequently transformed into an anteroposterior axis as the VE migrates anteriorly to form the anterior visceral endoderm (AVE). Both Nodal and Wnt signaling pathways are involved in these events. Loss of function in the Apc gene leads to constitutive ß-catenin activity that induces a proximalization of the epiblast with the activation of a subset of posterior mesendodermal genes, and loss of ability to induce the DVE. The loss of some DVE genes such as Hex and goosecoid is rescued in chimeras where only the epiblast was wild type; however, these DVE markers were no longer restricted distally but covered the entire epiblast. Thus, the Apc gene is needed in both embryonic and extraembryonic lineages for normal PD patterning around implantation, suggesting that early restricted activation of the Wnt pathway may be important for initiating axial asymmetries. In addition, it was found that nuclear ß-catenin and other molecular markers are asymmetrically expressed by 4.5 days (Chazaud, 2006).

Aberrant Wnt/beta-catenin signaling following loss of the tumor suppressor adenomatous polyposis coli (APC) is thought to initiate colon adenoma formation. Using zebrafish and human cells, it was shown that homozygous loss of APC causes failed intestinal cell differentiation but that this occurs in the absence of nuclear beta-catenin and increased intestinal cell proliferation. Therefore, loss of APC is insufficient for causing beta-catenin nuclear localization. APC mutation-induced intestinal differentiation defects instead depend on the transcriptional corepressor C-terminal binding protein-1 (CtBP1), whereas proliferation defects and nuclear accumulation of beta-catenin require the additional activation of KRAS (K-ras). These findings suggest that, following APC loss, CtBP1 contributes to adenoma initiation as a first step, whereas KRAS activation and beta-catenin nuclear localization promote adenoma progression to carcinomas as a second step. Consistent with this model, human familial adenomatous polyposis adenomas showed robust upregulation of CtBP1 in the absence of detectable nuclear beta-catenin, whereas nuclear beta-catenin was detected in carcinomas (Phelps, 2009).

Apc inhibition of Wnt signaling regulates supernumerary tooth formation during embryogenesis and throughout adulthood

The ablation of Apc function or the constitutive activation of beta-catenin in embryonic mouse oral epithelium results in supernumerary tooth formation, but the underlying mechanisms and whether adult tissues retain this potential are unknown. This study shows that supernumerary teeth can form from multiple regions of the jaw and that they are properly mineralized, vascularized, innervated and can start to form roots. Even adult dental tissues can form new teeth in response to either epithelial Apc loss-of-function or beta-catenin activation, and the effect of Apc deficiency is mediated by beta-catenin. The formation of supernumerary teeth via Apc loss-of-function is non-cell-autonomous. A small number of Apc-deficient cells is sufficient to induce surrounding wild-type epithelial and mesenchymal cells to participate in the formation of new teeth. Strikingly, Msx1, which is necessary for endogenous tooth development, is dispensable for supernumerary tooth formation. In addition, Fgf8, a known tooth initiation marker, was identified as a direct target of Wnt/beta-catenin signaling. These studies identify key mechanistic features responsible for supernumerary tooth formation (Wang, 2009).

Wnt signaling is active in developing teeth from the initiation stage, and continues throughout tooth differentiation. At the initiation of tooth development, Wnt4 and Wnt6 are expressed throughout oral and dental epithelium, whereas Wnt10a and Wnt10b transcripts are concentrated in the presumptive dental epithelium. Wnt3 and Wnt7b are not expressed in dental epithelium, but in the flanking oral epithelium. Wnt5a is the only Wnt signal reported to be expressed in the mesenchyme. Apc is an inhibitor of Wnt/β-catenin signaling, and in K14-Cre1Amc;Apccko/cko mice, β-catenin protein levels were significantly upregulated. Not surprisingly, therefore, the dental epithelial phenotypes in K14-Cre1Amc;Apccko/cko mice resemble those in mice with constitutive activation of β-catenin in the epithelium. However, both Apc and β-catenin have additional functions besides their interaction in the Wnt/β-catenin signaling pathway. Nonetheless, it was found that genetic depletion of β-catenin in K14-Cre1Amc;Apccko/cko mice blocked supernumerary tooth formation. This result formally proves that Apc acts to prevent supernumerary tooth formation by inhibiting Wnt/β-catenin signaling, and that the induction of supernumerary teeth by Apc loss-of-function is due to activation of the Wnt/β-catenin signaling pathway (Wang, 2009).

Embryonic and young mice formed supernumerary teeth continuously from multiple regions of the jaw, whereas in older adult mice, supernumerary teeth were mainly observed in regions around the incisors. The lack of supernumerary teeth in the molar regions of older adult mice might reflect the degeneration of the dental lamina and dental epithelial cells that normally occurs coincident with the eruption of the molar teeth, and the differentiation of remaining jaw epithelium into stratified epithelium and oral mucosa. The mouse incisor differs from these regions in that it contains epithelial stem cells in the central core of the cervical loop regions, and both the labial and lingual dental epithelia contain stem cells. Indeed, supernumerary teeth were formed from both the labial and lingual sides of the principal incisors. However, they were not only restricted to cervical loop regions. Some supernumerary teeth were located near the incisor tip, far from the cervical loop region, whereas others developed from among differentiating ameloblasts. Whether supernumerary teeth form from progenitor cells that migrate from the cervical loops, or whether dental progenitor cells also reside in other locations, requires further investigation (Wang, 2009).

APC interaction with beta-catenin

The 'ß-catenin destruction complex' is central to canonical Wnt/ß-catenin signaling. The scaffolding protein Axin and the tumor suppressor adenomatous polyposis coli protein (APC) are critical components of this complex, required for rapid ß-catenin turnover. The crystal structure of a complex between ß-catenin and the ß-catenin-binding domain of Axin (Axin-CBD) was determined. The Axin-CBD forms a helix that occupies the groove formed by the third and fourth armadillo repeats of ß-catenin and thus precludes the simultaneous binding of other ß-catenin partners in this region. Biochemical studies demonstrate that, when ß-catenin is phosphorylated, the 20-amino acid repeat region of APC competes with Axin for binding to ß-catenin. It is proposed that a key function of APC in the ß-catenin destruction complex is to remove phosphorylated ß-catenin product from the Axin/GSK-3ß active site (Xing, 2003).

In the canonical Wnt/ß-catenin pathway, ß-catenin mediates the transmission of a Wnt signal into the nucleus and the subsequent activation of target genes. In the absence of a Wnt signal, a cytoplasmic protein complex containing glycogen synthase kinase-3ß (GSK-3ß), the adenomatous polyposis coli protein (APC), and the scaffolding protein Axin, among others, catalyzes the phosphorylation of ß-catenin. This complex has been termed the 'ß-catenin destruction complex' because phosphorylation of ß-catenin targets it for degradation by the proteasome. When the pathway is active, binding of Wnt to its receptors leads to the inactivation of the destruction complex and a consequent accumulation of ß-catenin. The ß-catenin translocates to the nucleus, where it binds to DNA-binding proteins of the Tcf/LEF family. Together they turn on the transcription of Wnt-responsive genes. Although ß-catenin levels may also be regulated by other Axin-independent pathways, phosphorylation of ß-catenin by the ß-catenin destruction complex is the central regulatory step of the canonical Wnt/ß-catenin signaling pathway (Xing, 2003 and references therein).

In the ß-catenin destruction complex, GSK-3ß phosphorylates the critical residues in the N terminus of ß-catenin, contingent upon priming phosphorylation by casein kinase I (CKI). By itself, GSK-3ß does not efficiently phosphorylate ß-catenin; thus Axin plays a critical role in bringing GSK-3ß, CKIalpha, and ß-catenin together to efficiently promote the phosphorylation reaction. The importance of Axin in ß-catenin destruction is underscored by the presence of mutations in the human AXIN1 gene in certain human cancers that are associated with increased ß-catenin levels (Xing, 2003 and references therein).

Another essential component of the destruction complex is the tumor suppressor APC. Mutations of APC cause the elevation of cytoplasmic ß-catenin levels and are found in ~85% of colon cancers. The function of APC in the ß-catenin destruction complex is connected with Axin, because the overexpression of Axin in APC-mutant cancer cells is sufficient to down-regulate ß-catenin levels in these cells. APC contains repetitive ß-catenin interaction motifs, including three 15-amino acid repeats (or possibly four) and seven 20-amino acid repeats. It has been shown that APC plays a role in the transportation of ß-catenin from the nucleus to the cytoplasm, where ß-catenin is phosphorylated and degraded. Although it has also been proposed that APC may attenuate ß-catenin levels by recruiting ß-catenin to the ß-catenin destruction complex, it remains unclear how APC plays an essential role in the ß-catenin destruction complex (Xing, 2003 and references therein).

In addition to Axin, APC, GSK-3ß, and CKI, many other proteins, such as protein phosphatase 2A (PP2A), have also been found to play a role in the ß-catenin destruction complex. A central question now is how these proteins interact to form a molecular machine that efficiently phosphorylates and degrades ß-catenin. Specifically, a catalytic machine must be efficient in both substrate recruitment and product release. How does the ß-catenin destruction complex keep ß-catenin in the complex long enough to be phosphorylated, yet release it quickly enough to maintain the efficiency of phosphorylation (Xing, 2003)?

The crystal structure was determined of a complex between the armadillo repeat region of ß-catenin and the ß-catenin-binding domain of Axin, thus revealing the structural basis of the ß-catenin/Axin interaction. This structure suggests that Axin and the 20-amino acid repeat region of APC compete for binding to ß-catenin when they are both involved in the ß-catenin destruction complex. Biochemical studies clearly show that these regions do compete for binding, but only when the 20-amino acid region is phosphorylated. Based on these data, it is suggested that APC is required for both the recruitment of ß-catenin and the removal of the phosphorylated ß-catenin from the Axin/GSK-3ß active site, which explains the critical role of APC in ß-catenin turnover (Xing, 2003).

Inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates colorectal neoplasia. One of the biochemical activities associated with the APC protein is down-regulation of transcriptional activation mediated by beta-catenin and T cell transcription factor 4 (Tcf-4). The protein products of mutant APC genes present in colorectal tumors are found to be defective in this activity. Furthermore, colorectal tumors with intact APC genes are found to contain activating mutations of beta-catenin that alter functionally significant phosphorylation sites. These results indicate that regulation of beta-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or beta-catenin (Morin, 1997).

beta-catenin is a multifunctional protein found in three cell compartments: the plasma membrane, the cytoplasm and the nucleus. The cell has developed elaborate ways of regulating the level and localization of beta-catenin to assure its specific function in each compartment. One aspect of this regulation is inherent in the structural organization of beta-catenin itself; most of its protein-interacting motifs overlap so that interaction with one partner can block binding of another at the same time. Using recombinant proteins, it was found that E-cadherin and lymphocyte-enhancer factor-1 (LEF-1) form mutually exclusive complexes with beta-catenin; the association of beta-catenin with LEF-1 is competed out by the E-cadherin cytoplasmic domain. Similarly, LEF-1 and adenomatous polyposis coli (APC) form separate, mutually exclusive complexes with beta-catenin. In Wnt-1-transfected C57MG cells, free beta-catenin accumulates and is able to associate with LEF-1. The absence of E-cadherin in E-cadherin minus embryonic stem (ES) cells also leads to an accumulation of free beta-catenin and its association with LEF-1, thereby mimicking Wnt signaling. beta-catenin/LEF-1-mediated transactivation in these cells is antagonized by transient expression of wild-type E-cadherin, but not of E-cadherin lacking the beta-catenin binding site. The potent ability of E-cadherin to recruit beta-catenin to the cell membrane and prevent its nuclear localization and transactivation has also been demonstrated using SW480 colon carcinoma cells (Orsulic, 1999).

The adenomatous polyposis coli (APC) tumor-suppressor protein, together with Axin and GSK3, forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of ß-catenin by the proteasome. Siah-1, the human homolog of Drosophila Seven in absentia, is a p53-inducible mediator of cell cycle arrest, tumor suppression, and apoptosis. Siah-1 interacts with the carboxyl terminus of APC and promotes degradation of ß-catenin in mammalian cells. The ability of Siahß-1 to downregulate ß-catenin signaling was also demonstrated by hypodorsalization of Xenopus embryos. Unexpectedly, degradation of ß-catenin by Siah-1 is independent of GSK3ß-mediated phosphorylation and does not require the F box protein ß-TrCP. These results indicate that APC and Siahß-1 mediate a novel ß-catenin degradation pathway linking p53 activation to cell cycle control (Liu, 2001).

The adenomatous polyposis coli (APC) protein is inactivated in most colorectal tumours. APC loss is an early event in tumorigenesis, and causes an increase of nuclear ß-catenin and its transcriptional activity. This is thought to be the driving force for tumour progression. APC shuttles in and out of the nucleus, but the functional significance of this has been controversial. APC truncations have been shown to be nuclear in colorectal cancer cells and adenocarcinomas, and this correlates with loss of centrally located nuclear export signals. These signals confer efficient nuclear export as measured directly by fluorescence loss in photobleaching (FLIP), and they are critical for the function of APC in reducing the transcriptional activity of ß-catenin in complementation assays of APC mutant colorectal cancer cells. Importantly, targeting a functional APC construct to the nucleus causes a striking nuclear accumulation of ß-catenin without changing its transcriptional activity. This evidence indicates that the rate of nuclear export of APC, rather than its nuclear import or steady-state levels, determines the transcriptional activity of ß-catenin (Rosin-Arbesfeld, 2003).

In C. elegans, Wnt signaling regulates a number of asymmetric cell divisions. During telophase, WRM-1/beta-catenin localizes asymmetrically to the anterior cortex and the posterior daughter's nucleus. However, cortical WRM-1's functions are not known. This study used a membrane-targeted form of WRM-1 to show that cortical WRM-1 inhibits Wnt signaling and the nuclear localization of WRM-1. These functions are mediated by APR-1/APC, which regulates WRM-1 nuclear export. APR-1 as well as PRY-1/Axin and Dishevelled homologs localize asymmetrically to the cortex. These results suggest a model in which cortical WRM-1 recruits APR-1 to the anterior cortex before and during division, and the cortical APR-1 stimulates WRM-1 export from the anterior nucleus at telophase. Because beta-catenin and APC are localized to the cortex in many cell types in different species, these results suggest that these cortical proteins may regulate asymmetric divisions or Wnt signaling in other organisms as well (Mizumoto, 2007).

The results strongly suggest that cortical WRM-1/β-catenin inhibits its own nuclear localization during asymmetric cell division in C. elegans. This inhibitory role contrasts with that of nuclear WRM-1, which is a positive regulator of the Wnt/MAPK pathway, probably through its promotion of the nuclear export of POP-1/TCF. Thus, WRM-1 possesses dual and antagonistic functions in the Wnt/MAPK pathway. Because, in the apr-1(RNAi) animals, WRM-1 localized to both daughters at telophase with WRM-1 being at the anterior cortex, APR-1 probably mediates cortical WRM-1's effects on the localization of nuclear WRM-1. Thus, the data indicate a novel role for cortical β-catenin as a regulator of its asymmetric nuclear localization, mediated by APC during asymmetric cell divisions (Mizumoto, 2007).

The results show that Wnt proteins regulate the asymmetric cortical localization of components of the Wnt/MAPK pathway during postembryonic asymmetric cell divisions in C. elegans. In the absence of Wnts, LIN-17/Fz, APR-1/APC, PRY-1/Axin, WRM-1/β-catenin, and LIT-1/MAPK were localized symmetrically to the cortex. Wnt signals from the posterior side of the cell provide cues for polarization, leading to the posterior localization of Fz receptors, like LIN-17 in the T cell and MOM-5 in the Q and V5 cells. These localized and activated Fz receptors probably recruit the Dsh proteins to the posterior cortex, as has been shown in Drosophila and Xenopus. Although there is no experimental evidence for the involvement of Dsh, the cortical Dsh proteins may stimulate the disassembly of the complex containing PRY-1, APR-1, LIT-1, and WRM-1, releasing them from the cortex. The delay in the establishment of cortical asymmetry in pry-1 mutants suggests that PRY-1 probably functions redundantly in this process with one or more additional proteins, e.g., APR-1. This model seems consistent with previous observations in mammals and Drosophila that Wnt signals stimulate the dissociation of β-catenin from the destruction complex. In this scenario, the disassembly of the cortical multiprotein complex does not occur at the anterior cortex, which restricts WRM-1 and APR-1 to the anterior cortex prior to cell division. The detailed mechanism by which these proteins are targeted to the cortex is currently unknown. It does not seem to depend on the direct interaction between β-catenin and cadherin, because WRM-1 does not bind cadherin. Consistently, zygotic RNAi of hmr-1, which caused severe defects in embryonic morphogenesis, did not affect the cortical WRM-1 localization in larvae that escaped lethality. Nonetheless, WRM-1 appears to play a central role in this targeting mechanism, because, as was shown in this study, cortical WRM-1 affects the targeting of APR-1 and LIT-1 to the cortex (Mizumoto, 2007).

The results strongly suggest that APR-1 shuttles between the cytoplasm and nucleus and exports WRM-1 from both the anterior and posterior nuclei at telophase, when WRM-1 nuclear asymmetry is established. In addition, WRM-1 regulates the cortical localization of APR-1. Therefore, it is plausible that cortical WRM-1 regulates its own nuclear localization through cortical APR-1 at telophase. In this case, a remaining question is how cortical APR-1 regulates WRM-1 export. One possibility is that APR-1 functions at the cortex to regulate microtubules, given that in Drosophila and mammals APC localizes to the plus ends of microtubules to regulate their stability. APC is also known to move along microtubules toward the plus ends. In the anterior nucleus, the complex containing WRM-1 and APR-1 that exits from the nucleus may be efficiently loaded at the anterior perinuclear region onto microtubules stabilized by cortical WRM-1 and APR-1. This might result in the enhancement of nuclear export from the anterior nucleus at telophase. Further analyses will be necessary to elucidate the roles of microtubules and APR-1 in asymmetric cell divisions. Whatever the functions of cortical APR-1, the results indicate that APR-1 plays a central role in converting the polarity of mother cells to the asymmetries of the daughter nuclei (Mizumoto, 2007).

In contrast to the finding that apr-1 inhibits the Wnt/MAPK pathway in postembryonic cells, apr-1 is a positive regulator of the pathway in the EMS division. Consistent with this, in apr-1(RNAi) embryos, WRM-1 fails to localize to the nuclei of the EMS daughter cells, suggesting that apr-1 is required for the nuclear localization of WRM-1. Therefore, the molecular mechanisms of the Wnt/MAPK pathway may be different between the EMS and seam cells (Mizumoto, 2007).

Although WRM-1 has armadillo repeats like β-catenin, its sequence is quite diverged from β-catenin, and it does not bind to APR-1/APC or POP-1/TCF. Accordingly, the Wnt/MAPK pathway is sometimes referred to as a 'noncanonical' pathway. However, this study shows that WRM-1 colocalizes with APR-1 and PRY-1/Axin, and that its nuclear localization is negatively regulated by APR-1. Therefore, the pathway may be more relevant to the Wnt/β-catenin pathway in other organisms than previously thought, even if the stability of WRM-1 is not regulated by Wnt (Mizumoto, 2007).

In other organisms, β-catenin and APC localize to the cortex in a variety of cells. In contrast to the observation in C. elegans that cortical β-catenin inhibits Wnt signaling, it has been reported, in Xenopus and Drosophila, that overexpression of a membrane-tethered form of β-catenin activates Wnt signaling. Nevertheless, it has also been reported that overexpression of cadherin, which likely recruits β-catenin to the cortex, inhibits Wnt signaling. Such effects on Wnt signaling are explained by the sequestration of Wnt-signaling components: Axin or APC by membrane-tethered β-catenin, and β-catenin itself. Therefore, the physiological roles of cortical β-catenin in Wnt signaling remain elusive, and it is possible that cortical β-catenin directly inhibits Wnt signaling in other organisms (Mizumoto, 2007).

A function for β-catenin in cell polarity regulation has not been clearly demonstrated in other organisms. However, there are reports on the asymmetric cortical localization of Armadillo/β-catenin and APC in Drosophila neuroblasts that undergo asymmetric divisions (McCartney, 1999; Akong, 2002). Furthermore, APC localizes to the cortex asymmetrically along with β-catenin in germline stem cells and regulates their asymmetric divisions in Drosophila (Yamashita, 2003). Therefore, the function of β-catenin and APC as polarity regulators may be conserved in other organisms (Mizumoto, 2007).

Wnt signaling is involved in the maintenance of stem cells that undergo self-renewing asymmetric cell division. Although there is no evidence that Wnt signals regulate the polarities of stem cell divisions, inappropriate Wnt activation is often correlated with tumor formation, which may be caused by the failure of asymmetric cell divisions. In fact, this study showed that apr-1(RNAi) animals showed overproduction of the seam cells, probably due to defects in asymmetric cell divisions. The data suggest that cortical β-catenin may suppress stem cell tumorigenesis by regulating the polarities of asymmetric stem cell divisions and/or by repressing the nuclear accumulation of β-catenin. Further analyses may shed light on unidentified functions of β-catenin in cell polarity regulation and/or in the Wnt-signaling pathway in other organisms (Mizumoto, 2007).

Testing models of the APC tumor suppressor/β-catenin interaction reshapes our view of the destruction complex in Wnt signaling

The Wnt pathway is a conserved signal transduction pathway that contributes to normal development and adult homeostasis, but is also misregulated in human diseases such as cancer. The tumor suppressor Adenomatous Polyposis Coli (APC) is an essential negative regulator of Wnt signaling inactivated in over 80% of colorectal cancers. APC participates in a multi-protein 'destruction complex' that targets the proto-oncogene β-catenin for ubiquitin-mediated proteolysis; however, the mechanistic role of APC in the destruction complex remains unknown. Several models of APC function have recently been proposed, many of which have emphasized the importance of phosphorylation of high affinity β-catenin binding-sites (20 amino acid repeats; 20Rs) on APC. This study tested these models by generating a Drosophila APC2 mutant lacking all β-catenin binding 20Rs and performing functional studies in human colon cancer cell lines and Drosophila embryos. The results are inconsistent with current models, as it was found that β-catenin binding to the 20Rs of APC is not required for destruction complex activity. In addition, an APC2 mutant was generated lacking all β-catenin binding-sites (including the 15Rs), and a direct β-catenin/APC interaction was found to be also not essential for β-catenin destruction, although it increases destruction complex efficiency in certain developmental contexts. Overall, these findings support a model whereby β-catenin binding sites on APC do not provide a critical mechanistic function per se, but rather dock β-catenin in the destruction complex to increase the efficiency of β-catenin destruction. Furthermore, in Drosophila embryos expressing some APC2 mutant transgenes a separation of β-catenin destruction and Wg/Wnt signaling outputs was observed, and it is suggested that cytoplasmic retention of β-catenin likely accounts for this difference (Yamulla, 2004).

alpha-Catenin interacts with APC to regulate beta-catenin proteolysis and transcriptional repression of Wnt target genes

Mutation of the adenomatous polyposis coli (APC) tumor suppressor stabilizes beta-catenin and aberrantly reactivates Wnt/beta-catenin target genes in colon cancer. APC mutants in cancer frequently lack the conserved catenin inhibitory domain (CID), which is essential for beta-catenin proteolysis. This study shows that the APC CID interacts with alpha-catenin, a Hippo signaling regulator and heterodimeric partner of beta-catenin at cell:cell adherens junctions. Importantly, alpha-catenin promotes beta-catenin ubiquitylation and proteolysis by stabilizing its association with APC and protecting the phosphodegron. Moreover, beta-catenin ubiquitylation requires binding to alpha-catenin. Multidimensional protein identification technology (MudPIT) proteomics of multiple Wnt regulatory complexes reveals that alpha-catenin binds with beta-catenin to LEF-1/TCF DNA-binding proteins in Wnt3a signaling cells and recruits APC in a complex with the CtBP:CoREST:LSD1 histone H3K4 demethylase to regulate transcription and beta-catenin occupancy at Wnt target genes. Interestingly, tyrosine phosphorylation of alpha-catenin at Y177 disrupts binding to APC but not beta-catenin and prevents repression of Wnt target genes in transformed cells. Chromatin immunoprecipitation studies further show that alpha-catenin and APC are recruited with beta-catenin to Wnt response elements in human embryonic stem cells (hESCs). Knockdown of alpha-catenin in hESCs prevents the switch-off of Wnt/beta-catenin transcription and promotes endodermal differentiation. These findings indicate a role for alpha-catenin in the APC destruction complex and at Wnt target genes (Choi, 2013).

The APC tumor suppressor counteracts beta-catenin activation and H3K4 methylation at Wnt target genes

The APC tumor suppressor controls the stability and nuclear export of β-catenin (β-cat), a transcriptional coactivator of LEF-1/TCF HMG proteins in the Wnt/Wg signaling pathway. β-cat and APC have opposing actions at Wnt target genes in vivo. The β-cat C-terminal activation domain associates with TRRAP/TIP60 and mixed-lineage-leukemia (MLL1/MLL2) SET1-type chromatin-modifying complexes in vitro, and β-cat promotes H3K4 trimethylation at the c-Myc gene in vivo. H3K4 trimethylation in vivo requires prior ubiquitination of H2B, and ubiquitin is found necessary for transcription initiation on chromatin but not nonchromatin templates in vitro. Chromatin immunoprecipitation experiments reveal that β-cat recruits Pygopus, Bcl-9/Legless, and MLL/SET1-type complexes to the c-Myc enhancer together with the negative Wnt regulators, APC, and βTrCP. Interestingly, APC-mediated repression of c-Myc transcription in HT29-APC colorectal cancer cells is initiated by the transient binding of APC, βTrCP, and the CtBP corepressor to the c-Myc enhancer, followed by stable binding of the TLE-1 and HDAC1 corepressors. Moreover, nuclear CtBP physically associates with full-length APC, but not with mutant SW480 or HT29 APC proteins. It is concluded that, in addition to regulating the stability of β-cat, APC facilitates CtBP-mediated repression of Wnt target genes in normal, but not in colorectal cancer cells (Sierra, 2006).

The data presented here support a model in which the APC tumor suppressor functions directly to counteract β-cat-mediated transcription at Wnt target genes in vivo. This possibility was first suggested by the finding that full-length APC cycles on and off the c-Myc enhancer in conjunction with β-cat and associated coactivators in LiCl-treated C2C12 cells. In contrast, the enhancer complex appears to be stable and does not cycle in HT29 CRC cells, which contain a Class II APC mutant protein that is unable to degrade β-cat. Most strikingly, the binding of the full-length APC protein to the c-Myc gene in HT29-APC cells correlates with the rapid disassembly of the Wnt enhancer complex in vivo and the subsequent decline in steady-state c-Myc mRNA levels, both of which significantly precede the drop in β-cat protein levels that occurs as a result of proteolytic degradation in the cytoplasm. Thus, the effect of APC on c-Myc transcription appears to be immediate and direct, and may serve to coordinate the switch between the β-cat coactivator and TLE1 corepressor complexes (Sierra, 2006).

The β-cat enhancer complex includes the Wnt coactivators Pygopus and Bcl-9/Lgs, which control the retention of β-cat in the nucleus and may also function directly in transcription. The observation that APC can also regulate nuclear transport of β-cat raises the possibility that these factors may reside within a larger regulatory complex that chaperones β-cat in and out of the nucleus and mediates its release from the DNA. Indeed, sequential ChIP (re-ChIP) data indicate that the mutant APC in HT29 colorectal cancer cells exists in a stable complex with β-cat and LEF-1 at the active c-Myc gene. This finding is unexpected because β-cat cannot bind simultaneously to APC and LEF-1, and thus, if the full-length APC is part of a larger β-cat:LEF enhancer complex, it may interact with other subunits. Alternatively, the full-length APC and β-cat may exist in different complexes that rapidly exchange at the enhancer. The current data indicate that targeting is mediated by the N-terminal half of the APC protein, and that CtBP and βTrCP appear only in conjunction with the full-length APC protein. How APC is recruited to Wnt enhancers remains an open and important question (Sierra, 2006).

The ChIP experiments also suggest that APC-mediated inhibition of c-Myc transcription in HT29 cells occurs in two steps, initiated by transient binding of APC, βTrCP, CtBP, and YY1 to the enhancer, and followed by stable binding of the TLE-1 and HDAC1 corepressors. The transient recruitment of APC and CtBP, at the time when β-cat, Bcl-9, Pygo, and other Wnt enhancer factors leave the DNA, strongly suggests a role for these factors in the exchange of Wnt coactivator and corepressor complexes. In this respect it is interesting that CtBP was shown recently to associate with APC, both in vivo and in vitro. The results confirm a high-affinity interaction between CtBP and the full-length APC protein induced in HT29-APC cells, as well as with the native (full-length) APC protein in 293 cells. Consequently, APC may function to recruit CtBP to Wnt enhancers. Although both CtBP and TLE-1 are well-established corepressors of Wnt target genes, the different functions of the two types of corepressors remain unclear, and the ChIP data suggest that they act at distinct steps. Together, these data suggest that APC counteracts β-cat function in the nucleus, as well as in the cytoplasm, and may facilitate turnover of the enhancer complex at responsive genes by recruiting βTrCP and CtBP (Sierra, 2006).

The roles of APC and Axin derived from experimental and theoretical analysis of the Wnt pathway

Wnt signaling plays an important role in both oncogenesis and development. Activation of the Wnt pathway results in stabilization of the transcriptional coactivator ß-catenin. Recent studies have demonstrated that axin, which coordinates ß-catenin degradation, is itself degraded. Although the key molecules required for transducing a Wnt signal have been identified, a quantitative understanding of this pathway has been lacking. This study developed a mathematical model for the canonical Wnt pathway that describes the interactions among the core components: Wnt, Frizzled, Dishevelled, GSK3ß, APC, axin, ß-catenin, and TCF. Using a system of differential equations, the model incorporates the kinetics of protein-protein interactions, protein synthesis/degradation, and phosphorylation/dephosphorylation. Initially a reference state of kinetic, thermodynamic, and flux data was defined from experiments using Xenopus extracts. Predictions based on the analysis of the reference state were used iteratively to develop a more refined model from which the effects of prolonged and transient Wnt stimulation were analyzed on ß-catenin and axin turnover. Several unusual features of the Wnt pathway were predicted, some of which were tested experimentally. An insight from this model, which was confirmed experimentally, is that the two scaffold proteins axin and APC promote the formation of degradation complexes in very different ways. This study could also explain the importance of axin degradation in amplifying and sharpening the Wnt signal, and it was shown that the dependence of axin degradation on APC is an essential part of an unappreciated regulatory loop that prevents the accumulation of ß-catenin at decreased APC concentrations. By applying control analysis to the mathematical model, this study demonstrates the modular design, sensitivity, and robustness of the Wnt pathway and derives an explicit expression for tumor suppression and oncogenicity (Lee, 2003).

Theory and quantitation are mutually dependent activities. It would seem unlikely that one would go to the trouble to measure detailed kinetic quantities without a specific model to test, and it is equally unlikely that realistic models can be constructed without the constraints of quantitative experimental data. The intent in trying to reproduce a substantial part of the Wnt pathway in Xenopus egg extracts was to acquire the kind of detailed kinetic data required to build a realistic model. There are several unusual advantages to the extract system that contributed to this effort. The Xenopus egg extract is essentially neat cytoplasm; it reproduces the in vivo rate of β-catenin degradation and responds to known regulators as expected from in vivo experiments. Kinetic experiments with high time resolution are possible in this system, since a well-stirred extract is presumably synchronous in ways in which collections of cells may not be. In extracts it is possible to precisely set the level of components by depletion or addition. The direct output of the canonical Wnt pathway is an easily measured cytoplasmic event, the degradation of β-catenin. Thus, in this unusual system it is possible to acquire quantitative information about signaling pathways, not achievable in vivo. At the same time, these extracts have limitations. The receptor events were not considered, and it is likely that reactions at the plasma membrane contribute to dynamic features. Also, the analysis is incomplete, since there are other components of Wnt signaling, such as casein kinase Iδ, casein kinase Iɛ, and PAR1, as well as cross-talk from other pathways, that influence the behavior of the system. The multiple phosphorylation steps were oversimplified. A simple interconversion of the phosphorylated and unphosphorylated complex of axin, APC, and GSK3β was assumed, whereas in reality multiple phosphorylation states exist within the complex; the states may be random or sequential. The information needed to provide a much more specific model of phosphorylation interconversions is not available at this time, although the model could easily be extended. Finally, there is the question of what Wnt process are being studied. Events in the cytoplasm of unfertilized eggs are being examined. Though endowed with all of the core components of the Wnt pathway, the egg is, as far as is known, transcriptionally silent and not involved in Wnt signaling, though this system is active very soon in embryogenesis. Thus, there is no biological in vivo behavior with which to compare the in vitro behavior. Nevertheless, the basic core circuitry is intact and is presumably prepared for the early Wnt events in the embryo. All the properties of the egg extract system are very similar to that circuitry in vertebrate somatic cells (Lee, 2003).

To build a mathematical description of the Wnt signaling system, Lee (2003) started with the basic circuitry discerned from previous studies in Xenopus embryos and mammalian cells, whose similarity to the in vitro system they had already confirmed. A system of differential equations was derived that described the time-dependent variations of the system variables, i.e., the concentrations of the pathway components and their complexes. Parameters of the model are binding constants of proteins, rate constants of phosphorylations and dephosphorylations, rate constants of protein degradation, and rates of protein synthesis. Model reduction was achieved by considering conservation relations and by applying rapid equilibrium approximations for selected binding processes. Despite these simplifications, the model consists of a nonlinear system of differential equations whose solution requires the use of computers. Not all of these parameters were accessible to measurement. To circumvent this problem, not only kinetic parameters characterizing individual steps were used as primary inputs, but quantities that are more easily accessible from experiment, such as the overall flux of β-catenin degradation. This allowed rate constants to be derived as well as protein concentrations in a reference state, where there was no Wnt signal. This state serves as a starting point for predicting the system behavior during Wnt signaling as well as after experimental perturbations (Lee, 2003).

The basic model reproduced quantitatively the behavior of the reference state, including perturbations of this state achieved by varying the concentration of axin, GSK3β, and TCF. It also reproduced extensions of this to the signaling state. A wide variety of different sets of experimental data could be simulated by the same model, employing the same sets of kinetic parameters. This process was approached iteratively. For example, the early model did not include nonaxin-dependent degradation of β-catenin, but inclusion of this process improved the fit to the experimental data. More significantly, addition of this process had interesting biological implications, which are discussed (Lee, 2003).

In many ways, one of the most peculiar findings was the very low concentration of axin in the Xenopus extracts. Axin levels in other organisms may similarly be very low: Drosophila axin can be detected by Western blotting only following its immunoprecipitation. Although theoretical and experimental studies have shown that axin is inhibitory at high concentrations, both indicate that axin is not present at the optimal concentration for the highest rate of β-catenin degradation. Therefore, axin levels are not set for optimality of β-catenin degradation, but are presumably optimized for some other purpose. Theoretically, axin levels must be held below the very sharp threshold of Dsh inhibition. Experimentally, these thresholds, which blunt Wnt signaling, are observed but are not as sharp as expected, and this may indicate some other compensatory effects. These thresholds would limit axin concentration to well below 1 nM if activated Dsh were constrained to concentrations of below 1 μM. Under these circumstances, it can be expected that axin would never be found at concentrations approaching those of other Wnt pathway components (50-100 nM) (Lee, 2003).

The low concentration of axin relative to other components (such as GSK3β, Dsh, and APC) has another design feature potentially very general and important for the modularity of metazoan signaling pathways. Axin is a critical node point for controlling β-catenin levels, but it also interacts with components shared with several other important pathways. The interaction of these components with axin fluctuates due to Wnt signals (reflecting changes in binding as well as changes in axin levels), yet because the concentration of axin is so low, there will be no appreciable change in the overall levels of GSK3β, Dsh, or APC (all these components important in other pathways would otherwise be driven to fluctuate). The very low axin concentration thus isolates the Wnt pathway from perturbing other systems, a simple mechanism to achieve modularity. Other scaffold proteins may serve similar functions in other pathways. These insights follow from a very simple measurement of axin concentration and suggest the utility of measuring the levels of signaling pathway components in different cell types and circumstances. Since quantitative and kinetic features may be important in defining modules, it suggests that qualitative circuit diagrams of signal transduction may overlook very important design features. Modularity within the Wnt pathway can be defined by an extension of a summation theorem which argues that the steady state of an entire pathway would have control coefficients that added to zero. When the Wnt pathway is broken down to several subpathways, it is found that within these subpathways the control coefficients would sum to zero at steady state. While some of this subdivision is obvious (i.e., the kinase phosphatase module involving the phosporylation of APC and axin complexed to GSK3β), in other cases, such as the β-catenin module, it is much less obvious. Here the reactions include the phosphorylation of β-catenin in the APC/axin/GSK3β complex, the release and degradation of β-catenin, and the synthesis and nonaxin-dependent degradation of β-catenin. Balanced perturbation of these subpathways as a whole will not affect the overall flux of β-catenin degradation. It is not clear whether this concept of modularity might be extended usefully in two other directions: modularity in systems not at steady state, i.e., with transients, and estimates of linkage between pathways by some definition of nonzero summations expressing the degree of independence or modularity (Lee, 2003).

This paper marks one of the first extensions of metabolic control theory to signal transduction. Metabolism and signal transduction seem very different, the former involving the transfer of mass and the latter the transfer of information. In addition, metabolic pathways generally involve dedicated components and the specificity of interaction of substrates and enzymes is very high. Signaling pathways share many components; interactions are often weak. Metabolism, which has had a long history of quantitative study, was a natural field for the development of control theory, and this theory has been successful in converting the specific information about the behavior of enzymes in a pathway to the overall behavior of metabolic circuits. Control coefficients are useful measures of the impact of one process or quantity on another. In its application to metabolism, it allowed erroneous concepts, such as the notion of a rate-determining step, to be disposed. In signal transduction, control coefficients might play a similar role. Here they can be used to indicate quantitively the effects of a particular reaction on some other property, such as flux through the pathway or concentration of another component. By this definition, certain rate constants, such as the phosphorylation and dephosphorylation of APC and axin, have a major influence on the levels of β-catenin, while others, such as the degradation rate of phosphorylated β-catenin, have little effect. The sign and magnitude of these control coefficients give some indication what gene products could be oncogenes or tumor suppressors. By this criterion APC, GSK3β, and axin are potent tumor suppressors, whereas β-catenin is an oncogene. Dsh would be expected to exert only moderate oncogenic effects. Clearly the effects of certain gene products are dependent on context, including their rate of synthesis and steady-state concentration. As understanding of pathways improves, the effect of mutation or pharmacologic inhibition could be estimated quantitatively using control coefficients. The differences between cell types and organisms could be exploited to better predict mutagenic and pharmacologic impact on signal propagation (Lee, 2003).

Despite considerable progress in identifying components of the Wnt pathway, many important mechanistic details are still lacking. This analysis has shown that Dsh seems to act to prevent the phosphorylation of the axin/APC complex, not the phosphorylation of β-catenin. Dsh (complexed to the GSK-3 binding protein FRAT1 or GBP, which has no Drosophila homolog) does not seem to be a general GSK3β inhibitor, like Li+, but rather is focused on the two scaffolding proteins. This was apparent from the biphasic nature of both the theoretical and experimental curves, which suggests that Dsh inhibits the rephosphorylation of axin/APC, but still allows many cycles of β-catenin phosphorylation, ubiquitination, and degradation. This mechanism was further proven by a timing-of-addition experiment. It needs to be further confirmed and extended by looking specifically at individual phosphorylation sites on all the components of the complex. Another insight into the mechanisms of complex formation and control of β-catenin degradation concerns the inhibition of β-catenin degradation at concentrations of axin approaching those of other components. This suggests that axin binds APC, GSK3β, and β-catenin in random order. The axin concentration is limited by other factors; owing to the low concentration of axin, random binding is not likely ever to be a problem. The situation for APC seems very different. The concentration of APC is comparable to that of the other components. Overexpression studies show no inhibitory effects. These theoretical and experimental observations suggest that APC as a scaffold must be very different from axin as a scaffold. Most likely, APC binds components in an ordered manner (Lee, 2003).

APC plays a critical role in the Wnt signaling pathway by tightly regulating ß-catenin turnover and localization. The central region of APC is responsible for APC-ß-catenin interactions through its seven 20 amino acid (20aa) repeats and three 15 amino acid (15aa) repeats. Using isothermal titration calorimetry, the binding affinities were determined of ß-catenin with an APC 15aa repeat fragment and each of the seven 20aa repeats in both phosphorylated and unphosphorylated states. Despite sequence homology, different ß-catenin binding repeats of APC have dramatically different binding affinities with ß-catenin and thus may play different biological roles. The third 20aa repeat is by far the tightest binding site for ß-catenin among all the repeats. The fact that most APC mutations associated with colon cancers have lost the third 20aa repeat underlines the importance of APC-ß-catenin interaction in Wnt signaling and human diseases. For every 20aa repeat, phosphorylation dramatically increases its binding affinity for ß-catenin, suggesting phosphorylation has a critical regulatory role in APC function. In addition, CD and NMR studies demonstrate that the central region of APC is unstructured in the absence of ß-catenin and Axin, and suggest that ß-catenin may interact with each of the APC 15aa and 20aa repeats independently (Liu,, 2006).

APC interaction with Gsk-3

The APC tumor-suppressor protein associates with beta-catenin, a cell adhesion protein that is upregulated by the WNT1 oncogene. The effects of exogenous APC expression were examined on the distribution and amount of beta-catenin in a colorectal cancer cell containing only mutant APC. Expression of wild-type APC causes a pronounced reduction in total beta-catenin levels by eliminating an excessive supply of cytoplasmic beta-catenin indigenous to the SW480 colorectal cancer cell line. This reduction is due to an enhanced rate of beta-catenin protein degradation. Truncated mutant APC proteins, characteristic of those associated with cancer, lack this activity. Mutational analysis reveals that the central region of the APC protein, which is typically deleted or severely truncated in tumors, is responsible for the down-regulation of beta-catenin. These results suggest that the tumor-suppressor activity of mutant APC may be compromised due to a defect in its ability to regulate beta-catenin (Munemitsu, 1995).

Regulation of cell adhesion and cell signaling by beta-catenin occurs through a mechanism likely involving the targeted degradation of the protein. Deletional analysis was used to generate a beta-catenin refractory to rapid turnover These deletants were used to examine beta-catenin's effects on complexes containing either cadherin or the adenomatous polyposis coli (APC) protein. The amino-terminal deletion of beta-catenin results in a protein with increased stability, which acts in a dominant fashion with respect to wild-type beta-catenin. Constitutive expression in AtT20 cells of a beta-catenin lacking 89 N-terminal amino acids (deltaN89beta-catenin) results in severely reduced levels of the more labile wild-type beta-catenin. The mutant beta-catenin is expressed at endogenous levels but displaces the vast majority of wild-type beta-catenin associated with N-cadherin. The deltaN89beta-catenin accumulates on the APC protein to a level 10-times that of wild-type beta-catenin; it also recruits a kinase into the APC complex. The kinase is highly active toward APC in vitro and promotes a sodium dodecyl sulfate gel band shift that is also evident for endogenous APC from cells expressing the mutant beta-catenin. Unlike wild-type beta-catenin, which partitions solely as part of a high-molecular-weight complex, the deltaN89 mutant protein also fractionates as a stable monomer, indicating that it has escaped the requirement to associate with other proteins. That similar N-terminal mutants of beta-catenin have been implicated in cellular transformation suggests that their abnormal association with APC may, in part, be responsible for this phenotype (Munemitsu, 1996).

APC is mutated in most colon cancers. The APC protein binds to the cellular adhesion molecule beta-catenin, which is a mammalian homolog of Armadillo, a component of the Wingless signaling pathway in Drosophila development. When beta-catenin is present in excess, APC binds to another component of the Wingless pathway, glycogen synthase kinase 3beta (GSK3beta), a mammalian homolog of Drosophila Shaggy/Zeste white 3. APC is a good substrate for GSK3 beta in vitro, and the phosphorylation sites were mapped to the central region of APC. Binding of beta-catenin to this region is dependent on phosphorylation by GSK3 beta (Rubinfeld, 1996).

The mutation cluster region in the APC gene defines a region of approximately 660 bp, in which the vast majority of its somatic mutations are found. These mutations disrupt the polypeptide chain, typically eliminating five of the seven repeated sequences of 20 amino acids (aa), each in the central region of the APC protein. To examine the relationship between loss of this structure and loss of function, APC deletion mutants were constructed that progressively truncate the protein across the mutation cluster region. The mutants were tested for their association with beta-catenin and their ability to down-regulate it in SW480 cells. The binding of beta-catenin to APC fragments requires the inclusion of only a single 20-aa repeat sequence, whereas down-regulation requires the presence of at least three of these repeat sequences; those including the second repeat exhibit the highest activity. The mutation of three conserved serine residues in the second repeat greatly reduce the activity of an otherwise highly active APC fragment. Thus, the repeated 20-aa sequence is directly implicated in beta-catenin turnover. The elimination of at least five of these seven repeats due to somatic mutations suggests that loss of beta-catenin regulation by APC is selected for during tumor progression (Rubinfeld, 1997).

Axin: a scaffolding protein that interacts with APC and Gsk-3

Axin antagonizes the developmental effects of Wnt in vertebrates. Axin simultaneously binds two components of the Wnt pathway, beta-catenin and its negative regulator, glycogen synthase kinase-3beta. In mammalian cells, Axin inhibits Wnt-1 stimulation of beta-catenin/lymphoid enhancer factor 1-dependent transcription. Axin also blocks beta-catenin-mediated transcription in colon cancer cells that have a mutation in the adenomatous polyposis coli gene. These findings suggest that Axin, by forming a complex with beta-catenin and glycogen synthase kinase-3beta, can block signaling stimulated by Wnt or by adenomatous polyposis coli mutations (Sakanaka, 1998).

The regulators of the G protein signaling (RGS) domain of Axin, a negative regulator of the Wnt signaling pathway, make a complex with full-length adenomatous polyposis coli (APC) in COS, 293, and L cells but not with truncated APC in SW480 or DLD-1 cells. The RGS domain directly interacts with the region containing the 20-amino acid repeats but not with that containing the 15-amino acid repeats of APC, although both regions are known to bind to beta-catenin. In the region containing seven 20-amino acid repeats, the region containing the latter five repeats binds to the RGS domain of Axin. Axin and beta-catenin simultaneously interact with APC. Furthermore, Axin stimulates the degradation of beta-catenin in COS cells. Taken together with observations that Axin directly interacts with glycogen synthase kinase-3beta (GSK-3beta) and beta-catenin, and that Axin promotes the GSK-3beta-dependent phosphorylation of beta-catenin, these results suggest that Axin, APC, GSK-3beta, and beta-catenin make a tetrameric complex, resulting in the regulation of the stabilization of beta-catenin (Kishida, 1998).

Inactivation of the adenomatous polyposis coli (APC) tumor suppressor protein is responsible for both inherited and sporadic forms of colon cancer. Growth control by APC may relate to its ability to downregulate beta-catenin post-translationally. In cancer, mutations in APC ablate its ability to regulate beta-catenin, and mutations in beta-catenin prevent its downregulation by wild-type APC. Moreover, signaling by the protein product of the wnt-1 proto-oncogene upregulates beta-catenin and promotes tumorigenesis in mice. In a Xenopus developmental system, Wnt-1 signaling is inhibited by Axin, the product of the murine fused gene. This suggests a possible link between Axin, the Wnt-1 signaling components beta-catenin and glycogen synthase kinase 3 beta (GSK3 beta), and APC. Human Axin (hAxin) binds directly to beta-catenin, GSK3 beta, and APC in vitro, and the endogenous proteins are found in a complex in cells. Binding sites for Axin map to a region of APC that is typically deleted due to cancer-associated mutations in the APC gene. Overexpression of hAxin strongly promotes the downregulation of wild-type beta-catenin in colon cancer cells, whereas mutant oncogenic beta-catenin is unaffected. The downregulation is increased by deletion of the APC-binding domain from Axin, suggesting that APC may function to derepress Axin activity. In addition, hAxin dramatically facilitates the phosphorylation of APC and beta-catenin by GSK3 beta in vitro. It is concluded that Axin acts as a scaffold on which APC, beta-catenin and GSK3 beta assemble to coordinate the regulation of beta-catenin signaling (Hart, 1998).

Glycogen synthase kinase-3 (GSK-3) mediates epidermal growth factor, insulin and Wnt signals to various downstream events such as glycogen metabolism, gene expression, proliferation and differentiation. A GSK-3 beta-interacting protein has been isolated from a rat brain cDNA library using a yeast two-hybrid method. This protein consists of 832 amino acids and possesses Regulators of G-protein Signaling (RGS) and Dishevelled (Dsh), two homologous domains in its N- and C-terminal regions, respectively. The predicted amino acid sequence of this GSK-3beta-interacting protein shows 94% identity with mouse Axin, which recently has been identified as a negative regulator of the Wnt signaling pathway; therefore, this protein has been called rAxin (rat Axin). rAxin interacts directly with, and is phosphorylated by, GSK-3beta. rAxin also interacts directly with the armadillo repeats of beta-catenin. The binding site of rAxin for GSK-3beta is distinct from the beta-catenin-binding site, and these three proteins formed a ternary complex. Furthermore, rAxin promotes GSK-3beta-dependent phosphorylation of beta-catenin. These results suggest that rAxin negatively regulates the Wnt signaling pathway by interacting with GSK-3beta and beta-catenin and mediating the signal from GSK-3beta to beta-catenin (Ikeda, 1998).

Using a yeast two-hybrid method, a novel protein has been identified that interacts with glycogen synthase kinase 3beta (GSK-3beta) and has 44% amino acid identity with Axin, a negative regulator of the Wnt signaling pathway. This protein has been termed Axil, for Axin like. Like Axin, Axil ventralizes Xenopus embryos and inhibits Xwnt8-induced Xenopus axis duplication. Axil is phosphorylated by GSK-3beta. Axil binds not only to GSK-3beta but also to beta-catenin; the GSK-3beta-binding site of Axil is distinct from the beta-catenin-binding site. Axil enhances GSK-3beta-dependent phosphorylation of beta-catenin. These results indicate that Axil negatively regulates the Wnt signaling pathway by mediating the GSK-3beta-dependent phosphorylation of beta-catenin, thereby inhibiting axis formation (Yamamoto, 1998).

Control of the stability of beta-catenin is central in the Wnt signaling pathway. The protein Conductin, an Axin homolog, forms a complex with both beta-catenin and the tumor suppressor gene product adenomatous polyposis coli (APC). Conductin induces beta-catenin degradation, although conductin mutants are deficient in complex formation stabilize beta-catenin. Fragments of APC that contain a conductin-binding domain also block beta-catenin degradation. Thus, conductin is a component of the multiprotein complex that directs beta-catenin to degradation and is located downstream of APC. In Xenopus embryos, conductin interfers with wnt-induced axis formation (Behrens, 1998).

Mutations at the mouse Fused locus have pleiotropic developmental effects, including the formation of axial duplications in homozygous embryos. Mouse Fused is not to be confused with Drosophila fused, which mediates the Hedgehog signal to activate decapentaplegic. The product of the Fused locus, Axin, displays similarities to RGS (Regulators of G-protein Signaling) and Dishevelled proteins. Axin blocks the stimulation of the Wnt signaling pathway, regulating an early step in axis formation downstream of GSK-3, the mammalian homolog of Drosophila Shaggy. The Axin sequence homologous to the RGS domain is found between amino acids 213 and 338. A C-terminal 51 amino acid segment is 40% identical to a conserved sequence near the N terminus of Drosophila Dsh and its vertebrate homologs. Mutant Fused alleles that cause axial duplications disrupt the major mRNA, suggesting that Axin negatively regulates the response to an axis-inducing signal. Injection of Axin mRNA into Xenopus embryos inhibits dorsal axis formation by interfering with signaling through the Wnt pathway. Ventral injection of an Axin mRNA lacking the RGS domain induces an ectopic axis, apparently through a dominant-negative mechanism. Axin appears to negatively regulate signaling through the Wnt pathway, either at the level of GSK-3 or further downstream, based on its ability to block Xwnt8, Dishevelled, or dominant negative GSK-3 from ectopic axis formation in Xenopus embryos. Thus, Axin is a novel inhibitor of Wnt signaling and regulates an early step in embryonic axis formation in both mammals and amphibians (Zeng, 1997).

Axin has been identified as a regulator of vertebrate embryonic axis induction that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicate that Axin functions downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin; subsequent studies have shown that Axin is part of a complex including these two proteins and adenomatous polyposis coli (APC). The roles of different Axin domains were examined in the effects on axis formation and beta-catenin levels. The regulators of G-protein signaling domain (major APC-binding site) and GSK3beta-binding site are required, whereas the COOH-terminal sequences, including a protein phosphatase 2A binding site and the DIX domain, are not essential. Some forms of Axin lacking the beta-catenin binding site can still interact indirectly with beta-catenin and regulate beta-catenin levels and axis formation. Thus in normal embryonic cells, interaction with APC and GSK3beta is critical for the ability of Axin to regulate signaling via beta-catenin. Myc-tagged Axin is localized in a characteristic pattern of intracellular spots as well as at the plasma membrane. NH2-terminal sequences are required for targeting to either of these sites, whereas COOH-terminal sequences increase localization at the spots. Coexpression of hemagglutinin-tagged Dishevelled (Dsh) reveals strong colocalization with Axin, suggesting that Dsh can interact with the Axin/APC/GSK3/beta-catenin complex, and may thus modulate its activity (Fagotto, 1999).

Axin and the adenomatous polyposis coli (APC) tumor suppressor protein are components of the Wnt/Wingless growth factor signaling pathway. In the absence of Wnt signal, Axin and APC regulate cytoplasmic levels of the proto-oncogene beta-catenin through the formation of a large complex containing these three proteins, glycogen synthase kinase 3beta (GSK3beta) and several other proteins. Both Axin and APC are known to be critical for beta-catenin regulation, and human cancers result from truncations in APC that eliminate the Axin-binding site. A protease-resistant domain of Axin that contains the APC-binding site is a member of the regulators of G-protein signaling (RGS) superfamily. The crystal structures of this domain alone, and also in complex with an Axin-binding sequence from APC, reveal that the Axin-APC interaction occurs at a conserved groove on a face of the protein that is distinct from the G-protein interface of classical RGS proteins. The RGS-SAMP3 structure also provides insights into the conservation of Axin-binding repeats in diverse species. Previous work has identified sequences from Drosophila APC (dAPC) and APC2 (eAPC) that have homology to the SAMP repeats of vertebrate APC. However, the distant homology between these sequences and those of vertebrate SAMP repeats have made it difficult to identify these sequences unambiguously as Axin-binding sites in the absence of functional studies. The conservation of those residues that have been observed to make contacts in the RGS-SAMP3 complex supports the theory that three such sequences from dAPC and one from eAPC are Axin-binding sites. Thus the molecular interactions observed in the Axin-APC complex provide a rationale for the evolutionary conservation seen in both proteins (Spink, 2000).

The APC tumor suppressor protein plays a critical role in regulating cellular levels of the oncogene product ß-catenin. APC binds to ß-catenin through a series of homologous 15 and 20 amino acid repeats. The crystal structure of a 15 amino acid ß-catenin binding repeat has been determined from APC bound to the armadillo repeat region of ß-catenin. Although it lacks significant sequence homology, the N-terminal half of the repeat binds in a manner similar to portions of E-cadherin and XTcf3, but the remaining interactions are unique to APC. Evidence from structural, biochemical and sequence data is presented, which suggests that the 20 amino acid repeats can adopt two modes of binding to ß-catenin (Spink, 2001).

The homology between the N-terminal regions of the APC 15 and 20mer repeats suggests that the APC 20mers may bind to ß-catenin by a mechanism similar to that of APC-rA (the 15mer repeat A from APC) binding. Alignment of the core homology region of the 20mers with that of the 15mers results in a reasonable alignment with E-cadherin and XTcf3, even outside of the core homology region. Serines from the 20mer C-terminus (e.g. hAPC-1 Ser1276 and 1278) align with the first two phosphoserines seen in the E-cadherin structure (pSer684 and 686) and two glutamic acid residues from XTcf3 (Glu26 and 28). This alignment is consistent with data on the binding of an APC 20mer construct to a series of ß-catenin point mutants. The two mutations that eliminate APC 20mer binding (Lys345Ala and Trp383Ala) are in residues that interact with Glu1034 of APC-rA, at the C-terminus of the 15mer peptide (Spink, 2001).

In order to assess whether the 15 and 20mer repeats of APC bind at the same site on ß-catenin, whether the two classes of repeats could compete for binding to ß-catenin was tested. A construct containing 15mer repeat A (APC-fA) can compete with binding of a construct containing two 20mer repeats (APC-2,3). The efficiency of the observed competition (with significant reduction of APC-2,3 binding even at a 1:1 ratio with APC-fA), suggests an extensive overlap between the binding sites of the two proteins. However, it remains possible that the 15 and 20mer binding sites are distinct but overlapping, or that presentation of the binding repeats within the larger constructs may result in steric clashes, even if their binding sites do not overlap (Spink, 2001).

The theory that the core homology regions of the 15 and 20mer repeats bind at the same site differs from a proposed mechanism for 20mer binding derived from the ß-catenin- E-cadherin complex structure. In that structure, a Ser-Leu-Ser-Ser-Leu (SLSSL) sequence from E-cadherin binds to ß-catenin in a phosphorylation-dependent manner, with phosphoserines at consensus GSK3ß sites within and just N-terminal to the SLSSL sequence interacting with ß-catenin. An SLSSL sequence is conserved in the C-terminal region of the 20 amino acid ß-catenin binding repeats of APC, suggesting that this region of the 20 amino acid repeats can interact with the SLSSL binding region. This hypothesis is consistent with mutagenesis experiments, which show that the binding of ß-catenin to an APC construct containing both 15 and 20mer repeats is affected by mutations in the SLSSL binding site of ß-catenin. Since several hydrophobic residues from the SLSSL region of E-cadherin make contacts with ß-catenin, binding at the SLSSL site need not be entirely dependent upon phosphorylation. However, phosphorylation of the GSK3ß consensus sites in E-cadherin increases its affinity for ß-catenin 1000-fold. Likewise, GSK3ß phosphorylation of the 20 amino acid repeat region of APC has been shown to increase its affinity for ß-catenin (Spink, 2001).

Alignment of the SLSSL sequence of an APC 20mer (e.g. hAPC-1, residues 1278-82) with that from E-cadherin (residues 690-694) results in the alignment of a conserved 20mer serine (hAPC-1 Ser1272) with an upstream GSK3ß site in E-cadherin (pSer686). This serine corresponds to the last residue of the 20mer core homology region. However, E-cadherin pSer686 binds to ß-catenin >18 Å from the end of the core homology region-binding site (APC-rA Ser1028). Thus, it would be impossible for a single 20mer repeat to bind by both mechanisms simultaneously. Since the structural, sequence and mutagenesis data provide evidence for each binding mechanism it is suggested that the 20mers can adopt two distinct binding modes, and that phosphorylation may act as a switch between these modes (Spink, 2001).

What might be the role of two binding modes in ß-catenin degradation? Although the role of APC in the degradation complex is not fully understood, it is likely to include sequestration of ß-catenin from Lef/Tcf before degradation and presentation of ß-catenin for phosphorylation and degradation. The two binding modes could simply increase the number of potential binding sites, and hence the effective affinity between APC and ß-catenin. Alternatively, the two binding modes could have distinct roles; for example, the unphosphorylated 20mers, in conjunction with the 15mers, may mediate the initial binding and sequestering of ß-catenin, whereas subsequent phosphorylation of the 20mers and a switch in their mode of binding to ß-catenin could be important for efficient presentation of ß-catenin to GSK3ß. The existence and roles, if any, of these alternatives await further experimentation (Spink, 2001).

APC interaction with DLG

Continued: Apc-like Evolutionary homologs part 2/3 | part 3/3

Apc-like: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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