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PubMed2001

Siah-1 mediates a novel beta-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein.

The adenomatous polyposis coli (APC) tumor-suppressor protein, together with Axin and GSK3beta, forms a Wnt-regulated signaling complex that mediates phosphorylation-dependent degradation of beta-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. We have now found that Siah-1 interacts with the carboxyl terminus of APC and promotes degradation of beta-catenin in mammalian cells. The ability of Siah-1 to downregulate beta-catenin signaling was also demonstrated by hypodorsalization of Xenopus embryos. Unexpectedly, degradation of beta-catenin by Siah-1 was independent of GSK3beta-mediated phosphorylation and did not require the F box protein beta-TrCP. These results indicate that APC and Siah-1 mediate a novel beta-catenin degradation pathway linking p53 activation to cell cycle control.

Adenomatous Polyposis Coli Protein↗

PubMed2001

Cell density and phosphorylation control the subcellular localization of adenomatous polyposis coli protein.

Loss of functional adenomatous polyposis coli protein (APC) leads to uncontrolled proliferation of colonic epithelial cells, as evidenced by polyp formation, a prelude to carcinogenesis. As a tumor suppressor, APC targets the oncogene beta-catenin for proteasome-mediated cytoplasmic degradation. Recently, it was demonstrated that APC also interacts with nuclear beta-catenin, thereby reducing beta-catenin's activity as a transcription cofactor and enhancing its nuclear export. The first objective of this study was to analyze how cellular context affected APC distribution. We determined that cell density but not cell cycle influenced APC's subcellular distribution, with predominantly nuclear APC found in subconfluent MDCK and intestinal epithelial cells but both cytoplasmic and nuclear APC in superconfluent cells. Redistribution of APC protein did not depend on continual nuclear export. Focusing on the two defined nuclear localization signals in the C-terminal third of APC (NLS1(APC) and NLS2(APC)), we found that phosphorylation at the CK2 site increased and phosphorylation at the PKA site decreased NLS2(APC)-mediated nuclear translocation. Cell density-mediated redistribution of beta-galactosidase was achieved by fusion to NLS2(APC) but not to NLS1(APC). Both the CK2 and PKA sites were important for this density-mediated redistribution, and pharmacological agents that target CK2 and PKA instigated relocalization of endogenous APC. Our data provide evidence that physiological signals such as cell density regulate APC's nuclear distribution, with phosphorylation sites near NLS2(APC) being critical for this regulation.

Active Transport, Cell Nucleus↗

PubMed1997

Nuclear and cytoplasmic localizations of the adenomatous polyposis coli protein.

Mutation of the adenomatous polyposis coli (APC) gene is an early step in the initiation of colon cancer. Because the distribution pattern of a protein within the cell can provide important clues as to function, we have used a combination of immunofluorescence microscopy and biochemical fractionation to determine the location of APC protein in epithelial cells. Immunofluorescence microscopy placed full-length APC protein in both the nucleus and the cytoplasm. The nuclear APC protein was concentrated in discrete subnuclear regions, including nucleoli, whereas the cytoplasmic APC protein concentrated at the leading edge of migrating cells. Colocalization of APC protein with rRNA confirmed a nucleolar localization. These immunocytochemical findings have been supported by cell fractionation, which demonstrated that full-length APC protein was located in both the membrane/cytoskeletal and the nuclear fractions.

Adenomatous Polyposis Coli Protein↗

PubMed1999

The adenomatous polyposis coli protein.

Mutations in the adenomatous polyposis coli (APC) gene are associated with most colorectal cancers. The APC protein has been implicated in many aspects of tumour development. This article will discuss recent data suggesting that APC may have multiple functions in the cell. First, APC is a component of the Wnt signalling pathway; second, APC may have a role in cell migration; finally, APC may regulate proliferation and apoptosis.

Adenomatous Polyposis Coli Protein↗

PubMed2000

Phosphorylation near nuclear localization signal regulates nuclear import of adenomatous polyposis coli protein.

Mutation of the adenomatous polyposis coli (APC) gene is an early step in the development of colorectal carcinomas. APC protein is located in both the cytoplasm and the nucleus. The objective of this study was to define the nuclear localization signals (NLSs) in APC protein. APC contains two potential NLSs comprising amino acids 1767-1772 (NLS1(APC)) and 2048-2053 (NLS2(APC)). Both APC NLSs are well conserved among human, mouse, rat, and fly. NLS1(APC) and NLS2(APC) each were sufficient to target the cytoplasmic protein beta-galactosidase to the nucleus. Mutational analysis of APC demonstrated that both NLSs were necessary for optimal nuclear import of full-length APC protein. Alignment of NLS2(APC) with the simian virus 40 large T antigen NLS (NLS(SV40 T-ag)) revealed sequence similarity extending to adjacent phosphorylation sites. Changing a serine residue (Ser(2054)) to aspartic acid mutated the potential protein kinase A site adjacent to NLS2(APC), resulting in both inhibition of the NLS2(APC)-mediated nuclear import of a chimeric beta-galactosidase fusion protein and a reduction of full-length APC nuclear localization. Our data provide evidence that control of APC's nuclear import through phosphorylation is a potential mechanism for regulating APC's nuclear activity.

Adenomatous Polyposis Coli Protein↗

PubMed2002

Association and regulation of casein kinase 2 activity by adenomatous polyposis coli protein.

Mutations in the adenomatous polyposis coli (APC) gene are responsible for familial adenomatous polyposis coli and also sporadic colorectal cancer development. By using antibodies raised against the N-terminal region of APC protein, we have detected the variable masses of endogenous APC proteins in individual cell lines established from human colorectal carcinomas caused by nonsense mutations of the gene. Phosphorylation of immunoprecipitates of full-length and truncated APC were observed in in vitro kinase reaction, indicating association of APC with protein kinase activity. The kinase activity complexed with APC was sensitive to heparin and used GTP as phosphoryl donor, suggesting an involvement of casein kinase 2 (CK2). Both CK2alpha- and beta-subunits were found to associate with APC in immunoprecipitates as well as in pull-down assays, with preferential interaction of APC with tetrameric CK2 holoenzyme. In synchronized cell populations, the association of APC with CK2 was cell cycle dependent, with the highest association in G(2)/M. Unexpectedly, APC immunoprecipitates containing full-length APC protein inhibited CK2 in vitro, whereas immunoprecipitates of truncated APC had little effect. This was confirmed by using recombinant APC, and the inhibitory region was localized to the C terminus of APC between residues 2086 and 2394. Overexpression of this fragment in SW480 cells suppressed cell proliferation rates as well as tumorigenesis. These results demonstrate a previously uncharacterized functional interaction between the tumor suppressor protein APC and CK2 and suggest that growth-inhibitory effects of APC may be regulated by inhibition of CK2.

Adenomatous Polyposis Coli Protein↗

PubMed2004

Adenomatous polyposis coli proteins and cell adhesion.

Adenomatous polyposis coli (APC) is an important tumour suppressor in the mammalian intestinal epithelium. It binds to beta-catenin and its role as a tumour suppressor depends predominantly on its ability to downregulate soluble beta-catenin, a key effector of the Wnt signalling pathway. However, epithelial cells have a distinct subcellular pool of beta-catenin, or Drosophila Armadillo, which functions as a structural component of adherens junctions. Notably, APC proteins can be associated with these adherens junctions, and recent evidence points to a role for APC in cellular adhesion. Thus, APC--like beta-catenin/Armadillo--may have a dual role in Wnt signal transduction and in cellular adhesion, which could be relevant to its activity as a tumour suppressor.

Adenomatous Polyposis Coli Protein↗

PubMed2001

The use of coiled-coil interactions for the analysis and micropreparative isolation of adenomatous polyposis coli protein complexes.

The coiled coil is a common structural motif found both as the dominant structure in fibrous proteins and as an oligomerization domain in a variety of cytoskeletal and extracellular matrix proteins. Coiled-coils typically consist of two to four helices that are supercoiled around one another in either parallel or antiparallel orientations. In the past few years our knowledge of the structure and specificity of coiled coil interactions has increased, allowing the de novo design and preparation of coiled-coils with well-defined structure and specificity. Indeed, the design and synthesis of a peptide that binds specifically to a single coiled-coil-containing protein, adenomatous polyposis coli (APC) has been reported. We have optimized solid-phase synthesis techniques to produce a modified form of the anti-APC peptide that contains a biotin moiety specifically placed so as to allow selective orientation onto the surface of a biosensor or affinity support. These peptide surfaces have been used to both monitor and purify APC and APC complexes from cellular extracts.

Adenomatous Polyposis Coli Protein↗

PubMed2005

The threshold level of adenomatous polyposis coli protein for mouse intestinal tumorigenesis.

The adenomatous polyposis coli (APC) gene, whose mutations are responsible for familial adenomatous polyposis, is a major negative controller of the Wnt/beta-catenin pathway. To investigate the dose-dependent effects of APC protein in suppressing intestinal tumorigenesis, we constructed mutant mice carrying hypomorphic Apc alleles Apc(neoR) and Apc(neoF) whose expression levels were reduced to 20% and 10% of the wild type, respectively. Although both hypomorphic heterozygotes developed intestinal polyps, tumor multiplicities were much lower than that in Apc(Delta716) mice, heterozygotes of an Apc null allele. Like in Apc(Delta716) mice, loss of the wild-type Apc allele was confirmed for all polyps examined in the Apc(neoR) and Apc(neoF) mice. In the embryonic stem cells homozygous for these hypomorphic Apc alleles, the level of the APC protein was inversely correlated with both the beta-catenin accumulation and beta-catenin/T-cell factor transcriptional activity. These results suggest that the reduced APC protein level increases intestinal polyp multiplicity through quantitative stimulation of the beta-catenin/T-cell factor transcription. We further estimated the threshold of APC protein level that forms one polyp per mouse as approximately 15% of the wild type. These results also suggest therapeutic implications concerning Wnt signaling inhibitors.

Adenomatous Polyposis Coli Protein↗

PubMed2001

Axin-dependent phosphorylation of the adenomatous polyposis coli protein mediated by casein kinase 1epsilon.

Axin and the adenomatous polyposis coli protein (APC) interact to down-regulate the proto-oncogene beta-catenin. We show that transposition of an axin-binding site can confer beta-catenin regulatory activity to a fragment of APC normally lacking this activity. The fragment containing the axin-binding site also underwent hyperphosphorylation when coexpressed with axin. The phosphorylation did not require glycogen synthase kinase 3beta but instead required casein kinase 1epsilon, which bound directly to axin. Mutation of conserved serine residues in the beta-catenin regulatory motifs of APC interfered with both axin-dependent phosphorylation and phosphorylation by CKIepsilon and impaired the ability of APC to regulate beta-catenin. These results suggest that the axin-dependent phosphorylation of APC is mediated in part by CKIepsilon and is involved in the regulation of APC function.

Adenomatous Polyposis Coli Protein↗

PubMed1999

Apc1638T: a mouse model delineating critical domains of the adenomatous polyposis coli protein involved in tumorigenesis and development.

The adenomatous polyposis coli (APC) gene is considered as the true gatekeeper of colonic epithelial proliferation: It is mutated in the majority of colorectal tumors, and mutations occur at early stages of tumor development in mouse and man. These mutant proteins lack most of the seven 20-amino-acid repeats and all SAMP motifs that have been associated with down-regulation of intracellular beta-catenin levels. In addition, they lack the carboxy-terminal domains that bind to DLG, EB1, and microtubulin. APC also appears to be essential in development because homozygosity for mouse Apc mutations invariably results in early embryonic lethality. Here, we describe the generation of a mouse model carrying a targeted mutation at codon 1638 of the mouse Apc gene, Apc1638T, resulting in a truncated Apc protein encompassing three of the seven 20 amino acid repeats and one SAMP motif, but missing all of the carboxy-terminal domains thought to be associated with tumorigenesis. Surprisingly, homozygosity for the Apc1638T mutation is compatible with postnatal life. However, homozygous mutant animals are characterized by growth retardation, a reduced postnatal viability on the B6 genetic background, the absence of preputial glands, and the formation of nipple-associated cysts. Most importantly, Apc1638T/1638T animals that survive to adulthood are tumor free. Although the full complement of Apc1638T is sufficient for proper beta-catenin signaling, dosage reductions of the truncated protein result in increasingly severe defects in beta-catenin regulation. The SAMP motif retained in Apc1638T also appears to be important for this function as shown by analysis of the Apc1572T protein in which its targeted deletion results in a further reduction in the ability of properly controlling beta-catenin/Tcf signaling. These results indicate that the association with DLG, EB1, and microtubulin is less critical for the maintenance of homeostasis by APC than has been suggested previously, and that proper beta-catenin regulation by APC appears to be required for normal embryonic development and tumor suppression.

Adenomatous Polyposis Coli Protein↗

PubMed2001

The adenomatous polyposis coli protein: in the limelight out at the edge.

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial and sporadic colonic tumours. APC is best known for its role in regulating beta-catenin, an important mediator of cell adhesion and a transcriptional activator. However, recent studies indicate that APC has additional roles in cytoskeletal regulation. It binds to microtubules directly and indirectly. Furthermore, indirect connections between APC and the actin cytoskeleton have also been described. Here, we integrate recent information describing the association between APC and the cytoskeleton to illustrate how this multifaceted protein might link different cytoskeletal elements to each other and to cellular signaling pathways.

Adenomatous Polyposis Coli Protein↗

PubMed2001

A role for the Adenomatous Polyposis Coli protein in chromosome segregation.

Mutations in the Adenomatous Polyposis Coli (APC) gene are responsible for familial colon cancer and also occur in the early stages of sporadic colon cancer. APC functions in the Wnt signalling pathway to regulate the degradation of beta-catenin (reviewed in refs 1-3). APC also binds to and stabilizes microtubules in vivo and in vitro, localizes to clusters at the ends of microtubules near the plasma membrane of interphase cells, and is an important regulator of cytoskeletal function. Here we show that cells carrying a truncated APC gene (Min) are defective in chromosome segregation. Moreover, during mitosis, APC localizes to the ends of microtubules embedded in kinetochores and forms a complex with the checkpoint proteins Bub1 and Bub3. In vitro, APC is a high-affinity substrate for Bub kinases. Our data are consistent with a role for APC in kinetochore-microtubule attachment and suggest that truncations in APC that eliminate microtubule binding may contribute to chromosomal instability in cancer cells.

Adenomatous Polyposis Coli Protein↗

PubMed2006

Membrane localization of adenomatous polyposis coli protein at cellular protrusions: targeting sequences and regulation by beta-catenin.

Adenomatous polyposis coli protein (APC) translocates to, and stabilizes, the plus-ends of microtubules. In microtubule-dependent cellular protrusions, APC frequently accumulates in peripheral clusters at the basal membrane. APC targeting to membrane clusters is important for cell migration, but the localization mechanism is poorly understood. In this study, we performed deletion mapping and defined a minimal sequence (amino acids 1-2226) that efficiently targets APC to membrane clusters. This sequence lacks DLG-1 and EB1 binding sites, suggesting that these partners are not absolutely required for APC membrane targeting. A series of APC sequences were transiently expressed in cells and compared for their ability to compete endogenous APC at the membrane; potent inhibition of endogenous APC targeting was elicited by the Armadillo- (binds KAP3A, B56alpha, and ASEF) and beta-catenin-binding domains. The Armadillo domain was predicted to inhibit APC membrane localization through sequestration of the kinesin-KAP3A complex. The role of beta-catenin in APC membrane localization was unexpected but affirmed by overexpressing the APC binding sequence of beta-catenin, which similarly reduced APC membrane staining. Furthermore, we used RNA interference to show that loss of beta-catenin reduced APC at membrane clusters in migrating cells. In addition, we report that transiently expressed APC-yellow fluorescent protein co-localized with beta-catenin, KAP3A, EB1, and DLG-1 at membrane clusters, but only beta-catenin stimulated APC anchorage at the membrane. Our findings identify beta-catenin as a regulator of APC targeting to membrane clusters and link these two proteins to cell migration.

Adenomatous Polyposis Coli Protein↗

PubMed2003

Nuclear accumulation of full-length and truncated adenomatous polyposis coli protein in tumor cells depends on proliferation.

The adenomatous polyposis coli (APC) tumor suppressor is a nucleocytoplasmic protein. The nuclear accumulation of APC was recently found to vary depending on cell density, suggesting that putative APC function(s) in the nucleus is controlled by the establishment of cell contacts. We report here that the density-dependent redistribution of APC between nucleus and cytoplasm prevails in 6/6 thyroid and colorectal carcinoma cell lines. Moreover, mutated APC lacking known nuclear localization sequences had the similar distribution pattern as the full-length protein. APC invariably accumulated in the nuclei of Ki-67 expressing cells, but was largely cytoplasmic when cell cycle exit was induced by serum starvation or at high cell density. APC colocalized with beta-catenin in the nucleus only in one cell line (SW480). Also, APC maintained a predominantly nuclear position in early confluent states when cytoplasmic beta-catenin was recruited to newly formed adherens-like junctions. The results indicate that nuclear targeting of APC is driven by cell cycle entry rather than altered cell-cell contact. The ability of C-terminally truncated APC to accumulate in the nucleus suggests that nuclear import signals other than NLS1(APC) and NLS2(APC) are functionally important. Residual function(s) of N-terminal APC fragments in tumor cells carrying APC mutations might be beneficial to tumor growth and survival.

Adenomatous Polyposis Coli Protein↗

PubMed1995

Phosphorylation of the adenomatous polyposis coli protein and its possible regulatory effects in cells.

The adenomatous polyposis coli (APC) gene is etiologically associated with familial adenomatous polyposis and gastrointestinal malignancies, but its cellular function and role in tumorigenesis are unclear. Recent reports indicate that wild-type, but not mutant, APC gene product (APC) is associated with and promotes the assembly of cytoskeletal microtubules in vitro, suggesting that this mechanism has importance in tumor development. Because other microtubule-associated proteins (MAPs) undergo phosphorylation in their normal functioning, we postulated that APC is a phosphoprotein. HCT116 cells, containing full-length APC protein, were [32P]-prelabeled, and a 300-kDa band corresponding to phosphorylated APC was immunoprecipitated using each of three different anti-APC antibodies. High voltage electrophoresis of [32P]-labeled APC showed the presence of phospho-serine and phospho-threonine residues. Further immunoprecipitation analyses showed phosphorylation of i) full-length APC in human lymphoblastoid cells and ii) carboxyl-truncated APC in SW480 and DiFi colon carcinoma cells. Thus, APC is probably a phosphoprotein in normal and malignant tissues. We hypothesize a mechanism whereby phosphorylation of APC may play a regulatory role in its interaction with microtubules. This may involve phosphorylation of (Ser/Thr)-Pro amino acid motifs in APC's basic domain. We propose that deletion of this domain disrupts APC binding to microtubules, explaining how APC mutations are linked to cancer development.

Adenomatous Polyposis Coli Protein↗

PubMed2004

The adenomatous polyposis coli protein: the Achilles heel of the gut epithelium.

The Adenomatous Polyposis coli (APC) gene is mutated or lost in most colon cancers, and the APC protein has emerged as a multifunctional protein that is not only involved in the Wnt-regulated degradation of -catenin, but also regulates cytoskeletal proteins and thus plays a role in cell migration, cell adhesion, and mitosis. The gut epithelium is uniquely dependent on an intricate balance between a number of fundamental cellular processes including migration, differentiation, adhesion, apoptosis, and mitosis. In this review, I discuss the molecular mechanisms that govern the various functions of APC and their relationship to the role of APC in colon cancer.

Adenomatous Polyposis Coli Protein↗

PubMed2001

Binding of the adenomatous polyposis coli protein to microtubules increases microtubule stability and is regulated by GSK3 beta phosphorylation.

Truncation mutations in the adenomatous polyposis coli protein (APC) are responsible for familial polyposis, a form of inherited colon cancer. In addition to its role in mediating beta-catenin degradation in the Wnt signaling pathway, APC plays a role in regulating microtubules. This was suggested by its localization to the end of dynamic microtubules in actively migrating areas of cells and by the apparent correlation between the dissociation of APC from polymerizing microtubules and their subsequent depolymerization [1, 2]. The microtubule binding domain is deleted in the transforming mutations of APC [3, 4]; however, the direct effect of APC protein on microtubules has never been examined. Here we show that binding of APC to microtubules increases microtubule stability in vivo and in vitro. Deleting the previously identified microtubule binding site from the C-terminal domain of APC does not eliminate its binding to microtubules but decreases the ability of APC to stabilize them significantly. The interaction of APC with microtubules is decreased by phosphorylation of APC by GSK3 beta. These data confirm the hypothesis that APC is involved in stabilizing microtubule ends. They also suggest that binding of APC to microtubules is mediated by at least two distinct sites and is regulated by phosphorylation.

Adenomatous Polyposis Coli Protein↗