APC interaction with DLG

APC binds to DLG, the human homolog of the Drosophila Discs large tumor suppressor protein. This interaction requires the C-terminal region of APC and the PDZ domain repeat region of DLG. APC colocalizes with DLG at the lateral cytoplasm in rat colon epithelial cells and at the synapse in cultured hippocampal neurons. These results suggest that the APC-DLG complex may participate in regulation of both cell cycle progression and neuronal function (Matsumine, 1996).

Human scribble, a novel tumor suppressor identified as a target of high-risk HPV E6 for ubiquitin-mediated degradation, interacts with adenomatous polyposis coli

Human scribble (hScrib), human homolog of the Drosophila tumor suppressor Scribble, has been identified a substrate of human papillomavirus E6 oncoproteins for ubiquitin-mediated degradation dependent on ubiquitin-protein ligase E6AP. Human Scribble, classified as a LAP protein containing leucine-rich repeats and PDZ domains, interacts with E6 through its PDZ domains and C-terminal PDZ domain-binding motif of E6 protein. Interaction between human Discs Large (hDlg), a substrate of E6 for the ubiquitin-mediated degradation, and adenomatous polyposis coli (APC) has been shown. This study investigated whether hScrib and APC interact with each other in vitro and in vivo. Interaction between hScrib and APC is mediated by the PDZ domains 1 and 4 of hScrib and C-terminal PDZ domain-binding motif of APC. Human Scribble co-localizes with APC at the synaptic sites of hippocampal neuron and at the tip of membrane protrusion in the epithelial cell line. Interference of the interaction between hScrib and APC causes disruption of adherens junction. Knockdown of hScrib expression by RNAi disrupts localization of APC at the adherens junction. These data suggest that hScrib may participate in the hDlg-APC complex through its PDZ domains and regulate cell cycle and neural function by associating with APC (Takizawa, 2006).

APC interaction with EB1 proteins and microtubules

Defects in the APC gene occur frequently in patients with familial adenomatous polyposis coli and are associated with the progression of sporadic tumors of the colon and stomach. The subcellular location of adenomatous polyposis coli (APC) protein resulting from transient expression of full length and partial APC complementary DNAs was examined in epithelial cells. Immunofluorescent detection reveals an association of APC with cytoplasmic microtubules. Expression of partial complementary DNA constructs indicates that the carboxy-terminal region of the APC protein, typically deleted in cancers, is essential for this association. The same APC polypeptides that associate with microtubules in vivo also dramatically promote their assembly in vitro. These results suggest that wild-type APC protein binds to and affects the assembly of microtubules, whereas the mutants identified in tumors have lost this activity (Munemitsu, 1994).

The adenomatous polyposis coli protein (APC) is mutated in familial adenomatous polyposis patients as well as in sporadic colorectal tumors. In an attempt to further understand the function of APC, the subcellular localization of APC was examined. Wild-type and mutant forms of APC were expressed in mammalian cells and protein detected by immunofluorescence using monoclonal and polyclonal antibodies. Staining of wildtype APC protein reveals a filamentous network that extends throughout the cytoplasm and colocalizes with microtubules. In striking contrast, mutant APC protein gives a diffuse cytoplasmic staining pattern. Treatment with a microtubule depolymerizing agent, nocodazole, causes APC as well as tubulin to become diffusely cytoplasmic. In addition, immunoperoxidase staining of transfected APC protein followed by transmission electron microscopy reveals staining of microtubules. These results suggest that wild-type but not mutant APC protein may be associated with the microtubule cytoskeleton (Smith, 1994).

Mutations of the APC gene play a critical role in both sporadic and familial forms of colorectal cancer. The vast majority of these mutations result in the loss of the carboxyl terminus of the protein. To further elucidate the function of APC, cellular proteins that associate with its carboxyl terminus were sought. One million human cDNA clones were screened with the use of the interaction trap two-hybrid system; 67 clones were found to have a phenotype suggestive of an APC-interacting protein. Nucleotide sequence analysis reveals that 48 of these clones are derived from a single novel gene named EBI (see Drosophila Eb1). The association of APC and EB1 proteins was confirmed with in vitro binding assays. mAbs against EB1 were then produced and used to demonstrate the association of APC and EB1 in vivo. The EB1 gene is predicted to encode a 268-amino acid protein without significant homology to proteins with known function. However, searches of nucleotide databases have found evidence for at least two related human genes and a yeast homolog. This conservation suggests an essential function for EB1 that might provide clues to the mechanism through which APC suppresses colonic neoplasia (Su, 1995).

Mutations in the adenomatous polyposis coli (APC) gene are linked to polyp formation in familial and sporadic colon cancer, but the functions of the protein are not known. APC protein localizes mainly to clusters of puncta near the ends of microtubules that extend into actively migrating regions of epithelial cell membranes. This subcellular distribution of APC protein requires microtubules, but not actin filaments. APC protein-containing membranes are actively involved in cell migration in response to wounding epithelial monolayers, the addition of the motorgen hepatocyte growth factor, and during the formation of cell-cell contacts. In the intestine, APC protein levels increase at the crypt/villus boundary, where cell migration is crucial for enterocyte exit from the crypt and where cells accumulate during polyp formation that is linked to mutations in the microtubule-binding domain of APC protein. Together, these data indicate that APC protein has a role in directed cell migration (Nathke, 1996).

What function of APC protein is regulated by beta-catenin? It is noteworthy that in actively migrating epithelial cells, bundles of microtubules invade cell extentions and coalesce at clusters of APC protein that are localized at the leading edge of cell protrusions. In vitro, APC protein binds to and bundles microtubules; in transfected cells, exogenous APC protein codistributes along the length of microtubules. Addition of nocodazole to cells results in disruption of both microtubules and APC protein localization to the tips of membrane extensions and inhibition of direct cell migration. An interesting corollary to these observations is that during the formation of stable extensions in growth cone outgrowth, individual microtubules actively invade cell protrusions and are subsequently organized into bundles that stabilize the direction of migration. Formation of cell extensions during epithelial tubulogenesis may involve similar processes in which establishing and stabilizing the direction of migration involves the reorganization and stabilization of microtubules. It is suggested that beta-catenin regulates a function of APC protein in organizing microtubules that are required for the formation and/or stabilization of cell extensions during tubulogenesis (Pollack, 1997).

The tumor-suppressor protein APC binds to microtubules and promotes tubulin assembly. In vivo the endogenous APC protein is mainly localized at the end of microtubules that are involved in active cell migration. Since most tumor-specific APC gene mutations lead to the loss of the microtubule binding domain, this interaction is assumed to play a crucial role in tumorigenesis. An APC protein fragment (amino acids 2219-2580) within the C-terminal part is enough to bind to non-assembled tubulin with high affinity. The binding of APC to tubulin does not lead to an alteration of the intrinsic GTPase activity of the non-assembled tubulin. The APC protein induces the tubulin assembly in a fast reaction and below the critical assembly concentration of tubulin. The APC protein induces the bundling of the assembled microtubules in a concentration-dependent manner. Regarding its biochemical properties, the analysed APC protein fragment strikingly resembles the members of the microtubule-associated protein family tau. This analogy may prove useful in understanding the role of the APC protein in the suppression of tumorigenesis (Deka, 1998).

The evolutionarily conserved protein EB1 originally was identified by its physical association with the carboxyl-terminal portion of the adenomatous polyposis coli (APC) tumor suppressor protein. The subcellular localization of EB1 in epithelial cells was studied by using immunofluorescence and biochemical techniques. EB1 colocalizes both to cytoplasmic microtubules in interphase cells and to spindle microtubules during mitosis, with pronounced centrosome staining. The cytoskeletal array detected by anti-EB1 antibody is abolished by incubation of the cells with nocodazole, an agent that disrupts microtubules; upon drug removal, EB1 localizes to the microtubule-organizing center. Immunofluorescence analysis of SW480, a colon cancer cell line that expresses only carboxyl-terminal-deleted APC unable to interact with EB1, demonstrates that EB1 remains localized to the microtubule cytoskeleton, suggesting that this pattern of subcellular distribution is not mediated by its interaction with APC. In vitro cosedimentation with taxol-stabilized microtubules demonstrates that a significant fraction of EB1 associates with microtubules. Recent studies of the yeast EB1 homologs Mal3 and Bim1p have demonstrated that both proteins localize to microtubules and are important in vivo for microtubule function. These results demonstrate that EB1 is a novel component of the microtubule cytoskeleton in mammalian cells. Associating with the mitotic apparatus, EB1 may play a physiologic role connecting APC to cellular division, coordinating the control of normal growth and differentiation processes in the colonic epithelium (Berrueta, 1998).

Human EB1 is a highly conserved protein that binds to the carboxyl terminus of the human adenomatous polyposis coli (APC) tumor suppressor protein, a domain of APC that is commonly deleted in colorectal neoplasia. EB1 belongs to a family of microtubule-associated proteins that includes Schizosaccharomyces pombe Mal3 and Saccharomyces cerevisiae Bim1p. Bim1p appears to regulate the timing of cytokinesis as demonstrated by a genetic interaction with Act5, a component of the yeast dynactin complex. Whereas the predominant function of the dynactin complex in yeast appears to be in positioning the mitotic spindle, in animal cells, dynactin has been shown to function in diverse processes, including organelle transport, formation of the mitotic spindle, and perhaps cytokinesis. Human EB1 can be coprecipitated with p150(Glued), a member of the dynactin protein complex. EB1 has also been found associated with the intermediate chain of cytoplasmic dynein (CDIC) and with dynamitin (p50), another component of the dynactin complex, but not with dynein heavy chain, in a complex that sediments at approximately 5S in a sucrose density gradient. The association of EB1 with members of the dynactin complex is independent of APC and is preserved in the absence of an intact microtubule cytoskeleton. The molecular interaction of EB1 with members of the dynactin complex and with CDIC may be important for microtubule-based processes (Berrueta, 1999).

The characteristics of the adenomatous polyposis coli (APC) associated protein EB1 were examined in mammalian cells. By immunocytochemistry EB1 has been shown to be closely associated with the microtubule cytoskeleton throughout the cell cycle. In interphase cells EB1 is associated with microtubules along their full length but is often particularly concentrated at their tips. During early mitosis, EB1 is localized to separating centrosomes and associated microtubules, while at metaphase it is associated with the spindle poles and associated microtubules. During cytokinesis EB1 is strongly associated with the midbody microtubules. Treatment with nocodazole causes a diffuse redistribution of EB1 immunoreactivity, whereas treatment with cytochalasin D has no effect. Interestingly, treatment with taxol abolishes the EB1 association with microtubules. In nocodazole washout experiments, EB1 rapidly becomes associated with the centrosome and repolymerizing microtubules. In taxol wash-out experiments EB1 rapidly re-associates with the microtubule cytoskeleton, resembling untreated control cells within 10 min. Immunostaining of SW480 cells containing truncated APC incapable of interaction with EB1, shows that the association of EB1 with microtubules throughout the cell cycle is not dependent on EB1's association with APC. These results suggest a role for EB1 in the control of microtubule dynamics in mammalian cells (Morrison, 1999).

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 ß-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. The microtubule binding domain is deleted in the transforming mutations of APC; however, the direct effect of APC protein on microtubules has never been examined. 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 GSK3ß. 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 (Zumbrunn, 2001).

EB1, a parter of APC, is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homolog (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, dynamics of single microtubules in real time were visualized. Depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. These results reveal crucial roles for EB1 in mitosis, which is postulated to involve its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex (Rogers, 2002).

Little is known about how nerve growth factor (NGF) signaling controls the regulated assembly of microtubules that underlies axon growth. A tightly regulated and localized activation of phosphatidylinositol 3-kinase (PI3K) at the growth cone is essential for rapid axon growth induced by NGF. This spatially activated PI3K signaling is conveyed downstream through a localized inactivation of GSK-3ß. These two spatially coupled kinases control axon growth via regulation of a microtubule plus end binding protein, adenomatous polyposis coli (APC). These results demonstrate that NGF signals are transduced to the axon cytoskeleton via activation of a conserved cell polarity signaling pathway (Zhou. 2004).

Lysophosphatidic acid (LPA) stimulates Rho GTPase and its effector, the formin mDia, to capture and stabilize microtubules in fibroblasts. Whether mammalian EB1 and adenomatous polyposis coli (APC) function downstream of Rho-mDia in microtubule stabilization was investigated. A carboxy-terminal APC-binding fragment of EB1 (EB1-C) functions as a dominant-negative inhibitor of microtubule stabilization induced by LPA or active mDia. Knockdown of EB1 with small interfering RNAs also prevents microtubule stabilization. Expression of either full-length EB1 or APC, but not an APC-binding mutant of EB1, is sufficient to stabilize microtubules. Binding and localization studies showed that EB1, APC and mDia may form a complex at stable microtubule ends. Furthermore, EB1-C, but not an APC-binding mutant, inhibits fibroblast migration in an in vitro wounding assay. These results show an evolutionarily conserved pathway for microtubule capture, and suggest that mDia functions as a scaffold protein for EB1 and APC to stabilize microtubules and promote cell migration (Wen, 2004).

A microtubule network on the basal cortex of polarized epithelial cells consists of non-centrosomal microtubules of mixed polarity. This study investigated the proteins that are involved in organizing this network; end-binding protein 1 (EB1), APC and p150Glued - although all considered to be microtubule plus-end-binding proteins - are localized along the entire length of microtubules within the network, and at T-junctions between microtubules. The network shows microtubule behaviours that arise from physical interactions between microtubules, including microtubule plus-end stabilization on the sides of other microtubules, and sliding of microtubule ends along other microtubules. APC also localizes to the basal cortex. Microtubules grew over and paused at APC puncta; an in vitro reconstituted microtubule network overlaid APC puncta; and microtubule network reconstitution was inhibited by function-blocking APC antibodies. Thus, APC is a component of a cortical template that guides microtubule network formation (Reilein, 2005).

APC and the kinetochore-associated mitotic checkpoint

Adenomatous polyposis coli (APC) and End-binding protein 1 (EB1) localize to centrosomes independently of cytoplasmic microtubules (MTs) and purify with centrosomes from mammalian cell lines. Localization of EB1 to centrosomes is independent of its MT binding domain and is mediated by its C-terminus. Both APC and EB1 preferentially localize to the mother centriole and EB1 forms a cap at the end of the mother centriole that contains the subdistal appendages as defined by epsilon-tubulin localization. Like endogenous APC and EB1, fluorescent protein fusions of APC and EB1 localize preferentially to the mother centriole. Depletion of EB1 by RNA interference reduces MT minus-end anchoring at centrosomes and delays MT regrowth from centrosomes. In summary, these data indicate that APC and EB1 are functional components of mammalian centrosomes and that EB1 is important for anchoring cytoplasmic MT minus ends to the subdistal appendages of the mother centriole (Louie, 2004).

A cancer causing truncation in APC [APC(1-1450)] dominantly interferes with mitotic spindle function, suggesting APC regulates microtubule dynamics during mitosis. This study examined the possibility that APC mutants interfere with the function of EB1, a plus-end microtubule-binding protein that interacts with APC and is required for normal microtubule dynamics. It is shown that siRNA-mediated inhibition of APC, EB1, or APC and EB1 together give rise to similar defects in mitotic spindles and chromosome alignment without arresting cells in mitosis; in contrast inhibition of CLIP170 or LIS1 cause distinct spindle defects and mitotic arrest. APC(1-1450) acts as a dominant negative by forming a hetero-oligomer with the full-length APC and preventing it from interacting with EB1, consistent with a functional relationship between APC and EB1. Live-imaging of mitotic cells expressing EB1-GFP demonstrates that APC(1-1450) compromises the dynamics of EB1-comets, increasing the frequency of EB1-GFP pausing. Together these data provide novel insight into how APC may regulate mitotic spindle function and how errors in chromosome segregation are tolerated in tumor cells (Green, 2005).

EB1 proteins bind to microtubule ends where they act in concert with other components, including the adenomatous polyposis coli (APC) tumor suppressor, to regulate the microtubule filament system. EB1 is a stable dimer with a parallel coiled coil, and dimerization is essential for the formation of its C-terminal domain (EB1-C). The crystal structure of EB1-C reveals a highly conserved surface patch with a deep hydrophobic cavity at its center. EB1-C binds two copies of an APC-derived C-terminal peptide (C-APCp1) with equal 5 microM affinity. The conserved APC Ile2805-Pro2806 sequence motif serves as an anchor for the interaction of C-APCp1 with the hydrophobic cavity of EB1-C. Phosphorylation of the conserved Cdc2 site Ser2789-Lys2792 in C-APCp1 reduces binding four-fold, indicating that the interaction APC-EB1 is post-translationally regulated in cells. These findings provide a basis for understanding the dynamic crosstalk of EB1 proteins with their molecular targets in eukaryotic organisms (Honnappa, 2005; full text of article).

The correct formation of stable but dynamic links between chromosomes and spindle microtubules (MTs) is essential for accurate chromosome segregation. However, the molecular mechanisms by which kinetochores bind MTs and checkpoints monitor this binding remain poorly understood. In this paper, the functions of six kinetochore-bound MT-associated proteins (kMAPs) were examined using RNAi, live-cell microscopy and quantitative image analysis. RNAi-mediated depletion of two kMAPs, the APC protein and its binding partner, EB1 (a microtubule plus-end tracking protein), are unusual in affecting the movement and orientation of paired sister chromatids at the metaphase plate without perturbing kinetochore-MT attachment per se. Quantitative analysis shows that misorientation phenotypes in metaphase are uniform across chromatid pairs even though chromosomal loss (CIN) during anaphase is sporadic. However, errors in kinetochore function generated by APC or EB1 depletion are detected poorly if at all by the spindle checkpoint, even though they cause chromosome missegregation. It is proposed that impaired EB1 or APC function generates lesions invisible to the spindle checkpoint and thereby promotes low levels of CIN expected to fuel aneuploidy and possibly tumorigenesis (Draviam, 2006).

In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. A nonperturbing fluorescently labeled monoclonal antibody and labeled tubulin were comicroinjected to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and have a decreased catastrophe frequency compared with non-APC-decorated MTs. Endogenous APC associates briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, EB1-green fluorescent protein and endogenous APC were monitored concomitantly in living cells. Only a small fraction of EB1 colocalizes with APC at any one time. APC-deficient cells and EB1 small interfering RNA showed that EB1 and APC localized at MT ends independently. Depletion of EB1 does not change the growth-stabilizing effects of APC on MT plus ends. In addition, APC remains bound to MTs stabilized with low nocodazole, whereas EB1 does not. Thus, the association of endogenous APC with MT ends correlates directly with their increased growth stability, this can occur independently of its association with EB1, and APC and EB1 can associate with MT plus ends by distinct mechanisms (Kita, 2006; full text of article).

Adenomatous polyposis coli (APC) protein is a large tumor suppressor that is truncated in most colorectal cancers. The carboxyl-terminal third of APC protein mediates direct interactions with microtubules and the microtubule plus-end tracking protein EB1. In addition, APC has been localized to actin-rich regions of cells, but the mechanism and functional significance of this localization have remained unclear. This study shows that purified carboxyl-terminal basic domain of human APC protein (APC-basic) binds directly to and bundled actin filaments and associates with actin stress fibers in microinjected cells. Actin filaments and microtubules compete for binding to APC-basic, but APC-basic also can cross-link actin filaments and microtubules at specific concentrations, suggesting a possible role in cytoskeletal cross-talk. APC interactions with actin in vitro are inhibited by its ligand EB1, and co-microinjection of EB1 preventes APC association with stress fibers. Point mutations in EB1 that disrupt APC binding relieves the inhibition in vitro and restores APC localization to stress fibers in vivo, demonstrating that EB1-APC regulation is direct. Because tumor formation and metastasis involve coordinated changes in the actin and microtubule cytoskeletons, this novel function for APC and its regulation by EB1 may have direct implications for understanding the molecular basis of tumor suppression (Moseley, 2007).

The accurate segregation of chromosomes in mitosis requires the stable attachment of microtubules to kinetochores. The details of this complex and dynamic process are poorly understood. This study reports the interaction of a kinetochore-associated mitotic checkpoint kinase, BubR1, with two microtubule plus end-associated proteins, adenomatous polyposis coli (APC) and EB1, providing a potential link in stable kinetochore microtubule attachment. Using immunodepletion from and antibody addition to Xenopus laevis egg extracts, it was shown that BubR1 and its kinase activity are essential for positioning chromosomes at the metaphase plate. BubR1 associates with APC and EB1 in egg extracts, and the complex formation is necessary for metaphase chromosome alignment. Using purified components, BubR1 directly phosphorylates APC and forms a ternary complex with APC and microtubules. These findings support a model in which BubR1 kinase may directly regulate APC function involved in stable kinetochore microtubule attachment (Zhang, 2007).

APC interaction with KAP3

The tumor suppressor gene adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC is involved in the proteasome-mediated degradation of beta-catenin, through its interaction with beta-catenin, GSK-3 beta and Axin. APC also interacts with the microtubule cytoskeleton and has been localized to clusters near the distal ends of microtubules at the edges of migrating epithelial cells. Moreover, in Xenopus laevis epithelial cells, APC has been shown to move along microtubules and accumulate at their growing plus ends. However, the mechanism of APC accumulation and the nature of these APC clusters remain unknown. APC is shown to interact with the kinesin superfamily (KIF) 3A-KIF3B proteins, microtubule plus-end-directed motor proteins, through an association with the kinesin superfamily-associated protein 3 (KAP3: see Drosophila Kinesin associated protein 3). The interaction of APC with KAP3 was required for its accumulation in clusters, and mutant APCs derived from cancer cells were unable to accumulate efficiently in clusters. These results suggest that APC and beta-catenin are transported along microtubules by KAP3-KIF3A-KIF3B, accumulate in the tips of membrane protrusions, and may thus regulate cell migration (Jimbo, 2002).

APC sub-cellular location

The adenomatous polpyposis coli (APC) protein is mutated in most colorectal tumors. Nearly all APC mutations are truncations, and many of these terminate in the mutation cluster region located halfway through the protein. In cancer cells expressing mutant APC, beta-catenin is stabilized and translocates into the nucleus to act as a transcriptional co-activator of T-cell factor. During normal development, APC also promotes the destabilization of beta-catenin and Drosophila Armadillo. It does so by binding to the Axin complex which earmarks beta-catenin/Armadillo for degradation by the proteasome pathway. APC has a poorly understood regulatory role in this process. APC is shown in this study to contain highly conserved nuclear export signals 3' adjacent to the mutation cluster region that enable it to exit from the nucleus. This ability is lost in APC mutant cancer cells, and evidence is provided that beta-catenin accumulates in the nucleus as a result. Thus, the ability of APC to exit from the nucleus appears to be critical for its tumor suppressor function (Rosin-Arbesfeld, 2000).

Adenomatous polpyposis coli proteins are found in several subcellular compartments of mammalian and Drosophila cells including the cytoplasm, nucleus and adhesive cadherin/catenin junctions. To identify the targeting domains for these compartments, various fragments of the ubiquitously expressed Drosophila Adenomatous polyposis coli tumor suppressor homolog 2, called E-APC/dAPC2, were tagged with green fluorescent protein (GFP) and expressed in transgenic fly embryos and in monkey COS cells. The subcellular distribution of GFP-E-APC is indistinguishable from that of endogenous E-APC in embryos. In COS cells transfected with GFP-E-APC, green fluorescence is seen in the cytoplasm, concentrated at the plasma membrane, but also some in the nucleus. Unexpectedly, an amino-terminal fragment of E-APC (ARDcore) accumulates in the nucleus. Evidently, E-APC is capable of entering the nucleus by means of its N terminus. This N terminus spans the highly conserved Armadillo repeat domain (ARD). beta-catenin contains a closely related ARD that mediates its nuclear import independently of the Ran/Importin machinery (Rosin-Arbesfeld, 2000).

In contrast, carboxy-terminal fragments of E-APC (Cterm1 and 2) are efficiently excluded from the nucleus, more so than the full-length protein. Thus, the C terminus of E-APC either contains a cytoplasmic anchoring domain or an efficient nuclear export signal (NES). To distinguish between these possibilities, the GFP constructs were tested by treating transfected cells with leptomycin B (LMB), a highly specific drug that inhibits nuclear export by directly blocking the nuclear export receptor CRM1. Indeed, this results in even distribution of Cterm1 and Cterm2 throughout cytoplasm and nucleus. Full-length E-APC also accumulates to some extent in the nucleus after LMB treatment. Notably, endogenous E-APC is retained efficiently in nuclei of LMB-treated Drosophila embryos. These results indicate the presence of an NES in the C terminus of E-APC (Rosin-Arbesfeld, 2000).

Thus APC proteins contain highly conserved and functional nuclear export signals. Three lines of evidence implicate the ability of APC to exit from the nucleus in its tumor suppressor function: the sharp 3' border of APC truncation mutations, the nuclear accumulation of truncated APC in APC mutant cancer cells, and the compromised ability of NES-less APC to reduce nuclear beta-catenin in these cells. The nuclear export function of APC appears to be the 5'-most tumor suppressor function within the protein. The ability of APC to bind Axin to destabilize beta-catenin, which is also clearly critical for its tumor suppressor function, is encoded slightly downstream, and additional functions may reside in its C terminus (Rosin-Arbesfeld, 2000).

It has been proposed that APC may shuttle beta-catenin/Armadillo from the nucleus and cytoplasm to the junctional compartment where the Axin complex appears to be anchored. This work provides evidence for a putative shuttling function of APC, since the subcellular distribution of beta-catenin mirrors that of APC in wild-type and APC mutant cancer cells. However, the nuclear beta-catenin in the cancer cells may not simply be a consequence of the loss of APC-mediated export. Perhaps beta-catenin is positively trapped in the nuclei by the mutant APC. Nuclear trapping of beta-catenin by truncated APC may provide a mechanistic explanation for the striking mutation pattern observed in colorectal tumors (Rosin-Arbesfeld, 2000).

Adenomatous polyposis coli (APC) is mutated in most colorectal cancers. APC downregulates nuclear ß-catenin, which is thought to be critical for its tumor suppressor function. However, APC may have additional and separate functions at the cell periphery. Polarized MDCK and WIF-B hepatoma cells were examined and APC was found to be associated with their lateral plasma membranes. This depends on the actin cytoskeleton but not on microtubules, and drug wash-out experiments suggest that APC is delivered continuously to the plasma membrane by a dynamic actin-dependent process. In polarized MDCK cells, APC also clusters at microtubule tips in their basal-most regions. Microtubule depolymerization causes APC to relocalize from these tips to the plasma membrane, indicating two distinct peripheral APC pools that are in equilibrium with each other in these cells. Truncations of APC such as those found in APC mutant cancer cells can neither associate with the plasma membrane nor with microtubule tips. The ability of APC to reach the cell periphery may thus contribute to its tumor suppressor function in the intestinal epithelium (Rosin-Arbesfeld, 2001).

Perhaps the most interesting observation of this study was that nocodazole treatment of MDCK cells shifts APC from microtubule-dependent clusters to the plasma membrane. This illustrates that these two peripheral locations of APC differ in terms of their cytoskeletal requirements. It provided evidence for the existence of two distinct pools of APC that are in equilibrium with each other (Rosin-Arbesfeld, 2001).

What are the factors that determine the choice of APC between these peripheral pools? Correlative evidence suggests that this choice depends on whether a cell is freely motile or part of an epithelial tissue: the cells examined fall broadly into two groups, showing either predominantly microtubule-dependent APC clusters (motile MDCK and COS cells), or predominantly actin-dependent membrane-associated APC (polarized WIF-B and MDCK cells, colorectal cancer cells derived from the intestinal epithelium, and native Drosophila epithelia and tissues). It thus seems that, in motile cells, APC prefers a microtubule-dependent mode to reach peripheral sites, whereas in epithelial tissue, APC prefers an actin-dependent mode to reach the plasma membrane. Perhaps, microtubules are more suitable than actin filaments for fast and transient movement of APC over relatively large distances in motile cells. However, actin filaments may be suitable for continuous delivery of APC to adhesive plasma membranes, given that these filaments are permanently connected to the cadherin/catenin complexes in these membranes (Rosin-Arbesfeld, 2001).

APC as a target of cell cycle regulation

Mutations in the tumor suppressor gene APC invariably lead to the development of colorectal cancer. The vast majority of these mutations are nonsense or frameshifts resulting in nonfunctional, truncated APC protein products. Eleven cyclin-dependent kinase (CDK) consensus phosphorylation sites have been identified in the frequently deleted carboxyl-terminal region of APC; loss of these phosphorylation sites by mutation could therefore compromise the ability of APC to inhibit cell growth. This report demonstrates that immunoprecipitates of full-length, but not truncated, APC protein include a mitosis-specific kinase activity in vivo. Biochemical and Western analysis of these immunoprecipitates confirms the presence of the CDK p34(cdc2). APC is a substrate for recombinant human p34(cdc2)-cyclin B1. Modification of APC by p34(cdc2) implicates phosphorylation as a mechanism for regulating APC function via a link to the cell cycle (Trzepacz, 1997).

Although inappropriate activation of the Wnt/ß-catenin pathway has been implicated in the development of hepatocellular carcinoma (HCC), the role of this signaling in liver carcinogenesis remains unclear. To investigate this issue, a mutant mouse strain, Apc(lox/lox), was constructed in which exon 14 of the tumor-suppressor gene adenomatous polyposis coli (Apc) is flanked by loxP sequences. i.v. injection of adenovirus encoding Cre recombinase (AdCre) at high multiplicity inactivated the Apc gene in the liver and resulted in marked hepatomegaly, hepatocyte hyperplasia, and rapid mortality. ß-Catenin signaling activation was demonstrated by nuclear and cytoplasmic accumulation of ß-catenin in the hepatocytes and by the induction of ß-catenin target genes (glutamine synthetase, glutamate transporter 1, ornithine aminotransferase, and leukocyte cell-derived chemotaxin 2) in the liver. To test a long-term oncogenic effect, mice were inoculated with lower doses of AdCre, compatible with both survival and persistence of ß-catenin-activated cells. In these conditions, 67% of mice developed HCC. ß-Catenin signaling was strongly activated in these Apc-inactivated HCCs. The HCCs were well, moderately, or poorly differentiated. Indeed, their histological and molecular features mimicked human HCC. Thus, deletion of Apc in the liver provides a valuable model of human HCC, and, in this model, activation of the Wnt/ß-catenin pathway by invalidation of Apc is required for liver tumorigenesis (Colnot, 2004).

APC and apoptosis

APC protein is differentially expressed in the normal colonic crypt and believed to be involved in colonic cell maturation. An investigation was carried out to determine if expression of the APC protein is associated with cell death in colonic epithelial cells in vitro. Cells attached to flasks have a low frequency of apoptosis (1-3%), whereas cells that detach from flasks and float in the medium have a high proportion of apoptotic cells (36-96%, depending on the cell line). The full-length 300-kDa or truncated APC protein, normally expressed by the attached cells, is lost in the floating apoptotic cells in 8/11 colon tumour cell lines examined. Anti-APC antibody detects a 90-kDa protein in the floating apoptotic cells of all cell lines investigated, which was not present in the attached cells. Furthermore, loss of full-length APC and gain of the 90-kDa protein is observed in the apoptotic cells of 2 cell lines derived from other tissues: the SV40-transformed fibroblast cell line CMSV40fib and the lymphoblastoid B-cell line BJA-B. In cells repeatedly frozen and thawed, believed to induce necrotic cell death, full-length or truncated APC is also lost, though a 95-kDa protein is observed, distinct from that in apoptotic cells. Specific loss of full-length or truncated APC (resulting in a 90-kDa protein in apoptotic cells but a 95-kDa protein in necrotic cells) is therefore associated with cell death. These findings suggest a possible role for APC in cell survival (Browne, 1994).

Physiological regeneration of colonic epithelium entails proliferation at the crypt base and cell loss by shedding or cell death. The aim of this study was to localize and assess the rate of apoptosis in normal and neoplastic colonic epithelium with respect to zones of proliferation. Familial adenomatous polyposis (FAP) was chosen as a model to study neoplastic transformation of colonic mucosa at an early stage. By detection of genomic fragmentation, two different patterns of enterocytic apoptosis emerged: (1) apoptotic bodies being engulfed by adjacent epithelial cells, and (2) apoptotic cells with only subtle morphological changes being extruded into the gut lumen. The engulfment pattern is seen predominantly in the crypts of the normal colonic mucosa and, although very rare, is clearly confined to the basal proliferation compartment. The extrusion pattern is restricted to the luminal mucosal surface. Adenomas of FAP show highly increased numbers of apoptotic bodies, which are scattered throughout the transformed mucosa. Both patterns of apoptosis are topographically intermingled although the extrusion pattern prevailed at the luminal adenoma surfaces. Whereas cells in cycle are somewhat more numerous in the upper parts of the crypts, apoptosis occurs with increased frequency at sites beneath the proliferation maximum, suggesting an inverted direction for epithelial cell migration in adenomas. These results suggest two distinct routes toward enterocytic apoptosis in the colonic mucosa leading to engulfment or extrusion of dying cells. Adenomatous transformation of colon epithelium is associated with a considerable increase of the cellular turnover rate and with a severe disturbance of the microtopographical localization of birth and death of enterocytes (Strater, 1995).

Intestinal trefoil factor 3 (TFF3) is a member of the trefoil family of peptides, small molecules constitutively expressed in epithelial tissues, including the gastrointestinal tract. TFF3 has been shown to promote migration of intestinal epithelial cells in vitro and to enhance mucosal healing and epithelial restitution in vivo. The effect of recombinant TFF3 (rTFF3) stimulation was evaluated on the expression and cellular localization of the epithelial (E)-cadherin-catenin complex, a prime mediator of Ca2+ dependent cell-cell adhesion, and the adenomatous polyposis coli (APC)-catenin complex in HT29, HCT116, and SW480 colorectal carcinoma cell lines. Stimulation by rTFF3 leads to cell detachment and to a reduction in intercellular adhesion in HT29 and HCT116 cells. In both cell lines, E-cadherin expression is down-regulated. The expression of APC, alpha-catenin and beta-catenin also is decreased in HT29 cells, with a translocation of APC into the nucleus. No change in cell adhesion was detected in SW480 cells or in the expression of E-cadherin, the catenins, or APC. TFF3 induces DNA fragmentation and morphological changes characteristic of apoptosis in HT29. Tyrphostin, a competitive inhibitor of protein tyrosine kinases, inhibits the effects of TFF3. These results indicate that by perturbing the complexes between E-cadherin, beta-catenin, and associated proteins, TFF3 modulates epithelial cell adhesion, migration, and survival (Efstathiou, 1998).

APC's role in the Wingless pathway

Apc-like Evolutionary homologs part 3/3 | back to part 1/3

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

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