The third 20 amino acid repeat is the tightest binding site of APC for beta-catenin

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

Differences between the interaction of beta-catenin with non-phosphorylated and single-mimicked phosphorylated 20-amino acid residue repeats of the APC protein

The tumour suppressor protein adenomatous polyposis coli (APC) regulates the level and the intracellular localisation of the proto-oncoprotein beta-catenin. There are indications that a region comprising seven homologous 20-amino acid residue repeats within the APC protein is responsible for the interaction with beta-catenin and that the phosphorylation of conserved serine residues within these repeats increases the affinity for beta-catenin. We used biophysical methods to analyse the beta-catenin binding of single repeats or repeat combinations as non-phosphorylated or phosphorylated recombinant proteins. The non-phosphorylated repeats showed similar affinities, no matter whether they were tested as single recombinant repeats or in combination with neighbouring repeats. This result makes a cooperative influence between the repetitive motifs unlikely. The phosphorylation of the APC protein was mimicked by specific serine/aspartate mutations, which align to serine residues in the cytoplasmic beta-catenin binding domain of E-cadherin. Remarkably, the mimicked phosphorylation of a serine, which is not involved in beta-catenin interaction in the E-cadherin/beta-catenin complex, led to a significant increase in the APC affinity for beta-catenin. These results indicate structural differences between the E-cadherin/beta-catenin and the APC/beta-catenin complexes and provide quantitative evidence for the importance of the APC phosphorylation for its interaction with beta-catenin.     

Molecular mechanisms of beta-catenin recognition by adenomatous polyposis coli revealed by the structure of an APC-beta-catenin complex.

The adenomatous polyposis coli (APC) tumor suppressor protein plays a critical role in regulating cellular levels of the oncogene product beta-catenin. APC binds to beta-catenin through a series of homologous 15 and 20 amino acid repeats. We have determined the crystal structure of a 15 amino acid beta-catenin binding repeat from APC bound to the armadillo repeat region of beta-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. We discuss the implications of this new structure for the design of therapeutics, and present evidence from structural, biochemical and sequence data, which suggest that the 20 amino acid repeats can adopt two modes of binding to beta-catenin.     

Chemical dissection of the APC Repeat 3 multistep phosphorylation by the concerted action of protein kinases CK1 and GSK3

A crucial event in machinery controlled by Wnt signaling is the association of beta-catenin with the adenomatous polyposis coli (APC) protein, which is essential for the degradation of beta-catenin and requires the multiple phosphorylation of APC at six serines (1501, 1503, 1504, 1505, 1507, and 1510) within its repeat three (R3) region. Such a phosphorylation is believed to occur by the concerted action of two protein kinases, CK1 and GSK3, but its mechanistic aspects are a matter of conjecture. Here, by combining the usage of variably phosphorylated peptides reproducing the APC R3 region and Edman degradation assisted localization of residues phosphorylated by individual kinases, we show that the process is initiated by CK1, able to phosphorylate S1510 and S1505, both specified by non-canonical determinants. Phosphorylation of S1505 primes subsequent phosphorylation of S1501 by GSK3. In turn, phospho-S1501 triggers the hierarchical phosphorylation of S1504 and S1507 by CK1. Once phosphorylated, S1507 primes the phosphorylation of both S1510 and S1503 by CK1 and GSK3, respectively, thus completing all six phosphorylation steps. Our data also rule out the intervention of CK2 despite the presence of a potential CK2 phosphoacceptor site, S1510LDE, in the R3 repeat. S1510 is entirely unaffected by CK2, while it is readily phosphorylated even in the unprimed peptide by CK1delta but not by CK1gamma. This discloses a novel motif significantly different from non-canonical sequences phosphorylated by CK1 in other proteins, which appears to be specifically recognized by the delta isoform of CK1.     

Axin-independent phosphorylation of APC controls beta-catenin signaling via cytoplasmic retention of beta-catenin

It has been shown that accumulation of free beta-catenin leads to mobility shift of adenomatous polyposis coli (APC) protein and that Axin facilitates this process. Here we show that the beta-catenin-mediated mobility shift of APC is due to phosphorylation of two domains of APC by casein kinase 1epsilon/glycogen synthase kinase 3beta and unknown kinase(s), respectively. Interestingly, our results suggest that this process does not require Axin. The phosphorylated APC showed higher affinity to beta-catenin in vivo, and fragments of APC containing the phosphorylated domains can inhibit beta-catenin/Tcf-mediated reporter activity regardless of their ability to reduce the level of beta-catenin. From our data we propose a new role of APC: accumulation of excessive cytoplasmic beta-catenin induces phosphorylation of APC and the phosphorylated APC retains beta-catenin in cytoplasm to prevent excessive beta-catenin signaling. The retained beta-catenin in cytoplasm by APC may be down-regulated by Axin 2, which is induced by beta-catenin/Tcf signaling.     

Adenomatous polyposis coli (APC) differentially regulates beta-catenin phosphorylation and ubiquitination in colon cancer cells

Most colorectal cancers have mutations of the adenomatous polyposis coli (APC) gene or the beta-catenin gene that stabilize beta-catenin and activate beta-catenin target genes, leading ultimately to cancer. The molecular mechanisms of APC function in beta-catenin degradation are not completely known. APC binds beta-catenin and is involved in the Axin complex, suggesting that APC regulates beta-catenin phosphorylation. Some evidence also suggests that APC regulates beta-catenin nuclear export. Here, we examine the effects of APC mutations on beta-catenin phosphorylation, ubiquitination, and degradation in the colon cancer cell lines SW480, DLD-1, and HT29, each of which contains a different APC truncation. Although the current models suggest that beta-catenin phosphorylation should be inhibited by APC mutations, we detected significant beta-catenin phosphorylation in these cells. However, beta-catenin ubiquitination and degradation were inhibited in SW480 but not in DLD-1 and HT29 cells. The ubiquitination ofbeta-catenin in SW480 cells can be rescued by exogenous expression of APC. The APC domains required for beta-catenin ubiquitination were analyzed. Our results suggest that APC regulates beta-catenin phosphorylation and ubiquitination by distinct domains and by separate molecular mechanisms.     

E-cadherin and APC compete for the interaction with beta-catenin and the cytoskeleton

beta-Catenin is involved in the formation of adherens junctions of mammalian epithelia. It interacts with the cell adhesion molecule E- cadherin and also with the tumor suppressor gene product APC, and the Drosophila homologue of beta-catenin, armadillo, mediates morphogenetic signals. We demonstrate here that E-cadherin and APC directly compete for binding to the internal, armadillo-like repeats of beta-catenin; the NH2-terminal domain of beta-catenin mediates the interaction of the alternative E-cadherin and APC complexes to the cytoskeleton by binding to alpha-catenin. Plakoglobin (gamma-catenin), which is structurally related to beta-catenin, mediates identical interactions. We thus show that the APC tumor suppressor gene product forms strikingly similar associations as found in cell junctions and suggest that beta-catenin and plakoglobin are central regulators of cell adhesion, cytoskeletal interaction, and tumor suppression.     

Dephosphorylation of Cdc20 is required for its C-box-dependent activation of the APC/C

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.     

Functional Comparison of Human Adenomatous Polyposis Coli (APC) and APC-Like in Targeting Beta-Catenin for Degradation

Truncating mutations affect the adenomatous polyposis coli (APC) gene in most cases of colon cancer, resulting in the stabilization of β-catenin and uncontrolled cell proliferation. We show here that colon cancer cell lines express also the paralog APC-like (APCL or APC2). RNA interference revealed that it controls the level and/or the activity of β-catenin, but it is less efficient and binds less well to β-catenin than APC, thereby providing one explanation as to why the gene is not mutated in colon cancer. A further comparison indicates that APCL down-regulates the β-catenin level despite the lack of the 15R region known to be important in APC. To understand this discrepancy, we performed immunoprecipitation experiments that revealed that phosphorylated β-catenin displays a preference for binding to the 15 amino acid repeats (15R) rather than the first 20 amino acid repeat of APC. This suggests that the 15R region constitutes a gate connecting the steps of β-catenin phosphorylation and subsequent ubiquitination/degradation. Using RNA interference and domain swapping experiments, we show that APCL benefits from the 15R of truncated APC to target β-catenin for degradation, in a process likely involving heterodimerization of the two partners. Our data suggest that the functional complementation of APCL by APC constitutes a substantial facet of tumour development, because the truncating mutations of APC in colorectal tumours from familial adenomatous polyposis (FAP) patients are almost always selected for the retention of at least one 15R.     

Mitotic regulation of the APC activator proteins CDC20 and CDH1

The ordered activation of the ubiquitin protein ligase anaphase-promoting complex (APC) or cyclosome by CDC20 in metaphase and by CDH1 in telophase is essential for anaphase and for exit from mitosis, respectively. Here, we show that CDC20 can only bind to and activate the mitotically phosphorylated form of the Xenopus and the human APC in vitro. In contrast, the analysis of phosphorylated and nonphosphorylated forms of CDC20 suggests that CDC20 phosphorylation is neither sufficient nor required for APC activation. On the basis of these results and the observation that APC phosphorylation correlates with APC activation in vivo, we propose that mitotic APC phosphorylation is an important mechanism that controls the proper timing of APC(CDC20) activation. We further show that CDH1 is phosphorylated in vivo during S, G2, and M phase and that CDH1 levels fluctuate during the cell cycle. In vitro, phosphorylated CDH1 neither binds to nor activates the APC as efficiently as does nonphosphorylated CDH1. Nonphosphorylatable CDH1 mutants constitutively activate APC in vitro and in vivo, whereas mutants mimicking the phosphorylated form of CDH1 are constitutively inactive. These results suggest that mitotic kinases have antagonistic roles in regulating APC(CDC20) and APC(CDH1); the phosphorylation of APC subunits is required to allow APC activation by CDC20, whereas the phosphorylation of CDH1 prevents activation of the APC by CDH1. These mechanisms can explain the temporal order of APC activation by CDC20 and CDH1 and may help to ensure that exit from mitosis is not initiated before anaphase has occurred.     

Modeling the Effect of APC Truncation on Destruction Complex Function in Colorectal Cancer Cells

In colorectal cancer cells, APC, a tumor suppressor protein, is commonly expressed in truncated form. Truncation of APC is believed to disrupt degradation of β—catenin, which is regulated by a multiprotein complex called the destruction complex. The destruction complex comprises APC, Axin, β—catenin, serine/threonine kinases, and other proteins. The kinases and , which are recruited by Axin, mediate phosphorylation of β—catenin, which initiates its ubiquitination and proteosomal degradation. The mechanism of regulation of β—catenin degradation by the destruction complex and the role of truncation of APC in colorectal cancer are not entirely understood. Through formulation and analysis of a rule-based computational model, we investigated the regulation of β—catenin phosphorylation and degradation by APC and the effect of APC truncation on function of the destruction complex. The model integrates available mechanistic knowledge about site-specific interactions and phosphorylation of destruction complex components and is consistent with an array of published data. We find that the phosphorylated truncated form of APC can outcompete Axin for binding to β—catenin, provided that Axin is limiting, and thereby sequester β—catenin away from Axin and the Axin-recruited kinases and . Full-length APC also competes with Axin for binding to β—catenin; however, full-length APC is able, through its SAMP repeats, which bind Axin and which are missing in truncated oncogenic forms of APC, to bring β—catenin into indirect association with Axin and Axin-recruited kinases. Because our model indicates that the positive effects of truncated APC on β—catenin levels depend on phosphorylation of APC, at the first 20-amino acid repeat, and because phosphorylation of this site is mediated by , we suggest that is a potential target for therapeutic intervention in colorectal cancer. Specific inhibition of is predicted to limit binding of β—catenin to truncated APC and thereby to reverse the effect of APC truncation.     

Crystal structure of a beta-catenin/Tcf complex

The Wnt signaling pathway plays critical roles in embryonic development and tumorigenesis. Stimulation of the Wnt pathway results in the accumulation of a nuclear beta-catenin/Tcf complex, activating Wnt target genes. A crystal structure of beta-catenin bound to the beta-catenin binding domain of Tcf3 (Tcf3-CBD) has been determined. The Tcf3-CBD forms an elongated structure with three binding modules that runs antiparallel to beta-catenin along the positively charged groove formed by the armadillo repeats. Structure-based mutagenesis defines three sites in beta-catenin that are critical for binding the Tcf3-CBD and are differentially involved in binding APC, cadherin, and Axin. The structural and mutagenesis data reveal a potential target for molecular drug design studies.     

Destruction complex function in the Wnt signaling pathway of Drosophila requires multiple interactions between Adenomatous polyposis coli 2 and Armadillo

The tumor suppressor Adenomatous polyposis coli (APC) negatively regulates Wnt signaling through its activity in the destruction complex. APC binds directly to the main effector of the pathway, β-catenin (βcat, Drosophila Armadillo), and helps to target it for degradation. In vitro studies demonstrated that a nonphosphorylated 20-amino-acid repeat (20R) of APC binds to βcat through the N-terminal extended region of a 20R. When phosphorylated, the phospho-region of an APC 20R also binds βcat and the affinity is significantly increased. These distinct APC-βcat interactions suggest different models for the sequential steps of destruction complex activity. However, the in vivo role of 20R phosphorylation and extended region interactions has not been rigorously tested. Here we investigated the functional role of these molecular interactions by making targeted mutations in Drosophila melanogaster APC2 that disrupt phosphorylation and extended region interactions and deletion mutants missing the Armadillo binding repeats. We tested the ability of these mutants to regulate Wnt signaling in APC2 null and in APC2 APC1 double-null embryos. Overall, our in vivo data support the role of phosphorylation and extended region interactions in APC2's destruction complex function, but suggest that the extended region plays a more significant functional role. Furthermore, we show that the Drosophila 20Rs with homology to the vertebrate APC repeats that have the highest affinity for βcat are functionally dispensable, contrary to biochemical predictions. Finally, for some mutants, destruction complex function was dependent on APC1, suggesting that APC2 and APC1 may act cooperatively in the destruction complex.     

Interaction among GSK-3, GBP, axin, and APC in Xenopus axis specification

Glycogen synthase kinase 3 (GSK-3) is a constitutively active kinase that negatively regulates its substrates, one of which is beta-catenin, a downstream effector of the Wnt signaling pathway that is required for dorsal-ventral axis specification in the Xenopus embryo. GSK-3 activity is regulated through the opposing activities of multiple proteins. Axin, GSK-3, and beta-catenin form a complex that promotes the GSK-3-mediated phosphorylation and subsequent degradation of beta-catenin. Adenomatous polyposis coli (APC) joins the complex and downregulates beta-catenin in mammalian cells, but its role in Xenopus is less clear. In contrast, GBP, which is required for axis formation in Xenopus, binds and inhibits GSK-3. We show here that GSK-3 binding protein (GBP) inhibits GSK-3, in part, by preventing Axin from binding GSK-3. Similarly, we present evidence that a dominant-negative GSK-3 mutant, which causes the same effects as GBP, keeps endogenous GSK-3 from binding to Axin. We show that GBP also functions by preventing the GSK-3-mediated phosphorylation of a protein substrate without eliminating its catalytic activity. Finally, we show that the previously demonstrated axis-inducing property of overexpressed APC is attributable to its ability to stabilize cytoplasmic beta-catenin levels, demonstrating that APC is impinging upon the canonical Wnt pathway in this model system. These results contribute to our growing understanding of how GSK-3 regulation in the early embryo leads to regional differences in beta-catenin levels and establishment of the dorsal axis.