Characterization of a 60S complex of the adenomatous polyposis coli tumor suppressor protein

The tumor suppressor protein adenomatous polyposis coli (APC) is a multifunctional protein with a well characterized role in the Wnt signal transduction pathway and roles in cytoskeletal regulation and cell polarity. The soluble pool of APC protein in colon epithelial tumor cells exists in two distinct complexes fractionating at approximately 20S and approximately 60S in size. The 20S complex contains components of the beta-catenin destruction complex and probably functions in the Wnt pathway. In this study, we characterized the molecular nature of the 60S APC- containing complex by examining known potential binding partners of APC. 60S APC did not contain EB1 or diaphanous, proteins that have been reported to interact with APC and are implicated in microtubule plus end stabilization. Nor did the two other microtubule associated proteins, MAP4 or KAP3, which is thought to link APC to kinesin motor proteins, associate with the 60S complex. Minor fractions of alpha-tubulin, gamma-tubulin and IQGAP1, a Rac1 and CDC42 effector that interacts with APC, specifically associated with APC in the 60S fraction. We propose that 60S APC is a discrete high molecular weight complex with a novel function in cytoskeletal regulation in epithelial cells apart from its well established role in targeting catenin destruction or its proposed role in microtubule plus end stabilization.     

The adenomatous polyposis coli (APC) tumor suppressor

Defects in the APC gene are inarguably linked to the progression of colon cancers that arise both sporadically and through the transmission of germline mutations. Genetic evidence from humans and mouse models suggest that APC is a classic tumor suppressor in that both alleles likely require inactivation for tumor growth to ensue. Nearly all of the mutations, germline and somatic, result in premature termination of the single polypeptide chain, normally consisting of 2843 amino acids. Several definable motifs have now been mapped to the linear amino acid sequence of the APC polypeptide. These include an oligomerization domain, armadillo repeats, binding sites for β-catenin, the human discs large protein, microtubules, and other proteins of unknown function. Inactivation of APC in cancer is likely due to loss of function(s) normally associated with the deleted protein structure.     

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 associates with the microtubule-destabilizing protein XMCAK

During cell division, the proper formation of a bipolar spindle and its function to segregate chromosomes requires precise coordination of microtubule-stabilizing and destabilizing activities. Globally destabilized, dynamic microtubules radiating from duplicated centrosomes are locally regulated by chromosomes. Proteins at the kinetochore of each sister chromatid mediate a dynamic attachment, allowing chromosome movement coupled to microtubule polymerization/depolymerization and error-correction mechanisms for improperly attached chromosomes. The tumor suppressor protein adenomatous polyposis coli (APC) stabilizes microtubules both in vitro and in vivo and is implicated in mitosis, although its mechanisms of action are not well characterized. Here, we show that in mitotic Xenopus egg extracts, the carboxyl-terminus of APC can associate with the amino terminus of the microtubule-destabilizing KinI, Xenopus mitotic centromere-associated kinesin (XMCAK), in a cytoplasmic complex. We find that like XMCAK, APC can localize to the centromere as well as the kinetochore region of mitotic chromosomes and does not require microtubules for chromosomal targeting in Xenopus egg extracts. We propose that the presence of these proteins in a complex brings together both positive and negative microtubule effectors, whose opposing activities may be regulated by additional factors, thereby providing precise control of both global and local microtubule dynamics.     

Ca regulates the subcellular localization of adenomatous polyposis coli tumor suppressor protein

Microtubule (MT) plus-end tracking proteins (+TIPs) are involved in the regulation of MT plus-end dynamics and stabilization. It was reported previously that an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by disruption of the plasma membrane stimulates rearrangement of MTs [T. Togo, Disruption of the plasma membrane stimulates rearrangement of microtubules and lipid traffic toward the wound site, J. Cell Sci. 119 (2006) 2780-2786], suggesting that some +TIPs are regulated by Ca²⁺. In the present study, the behavior of adenomatous polyposis coli (APC) following an increase in [Ca²⁺]_i was observed using Xenopus A6 epithelial cell expressing GFP-tagged APC. An increase in [Ca²⁺]_i by cell membrane disruption or by ionomycin treatment induced dissociation of APC without depolymerizing MTs. Inhibition of a tyrosine kinase and GSK-3β suppressed APC dissociation upon an increase in [Ca²⁺]_i. Western blotting analysis showed that Ca²⁺ transients activated GSK-3β through a tyrosine kinase. These results suggest that Ca²⁺ stimulates redistribution of APC through a tyrosine kinase- and GSK-3β-dependent pathway.     

MicroRNA-122a functions as a novel tumor suppressor downstream of adenomatous polyposis coli in gastrointestinal cancers

Aberrant regulation of APC/β-catenin signaling pathway is common in the pathogenesis of colorectal and other cancers. Targets regulated by APC/β-catenin signaling pathway play crucial roles in cancer development. In the current study, we aimed to illustrate the influence of APC/β-catenin signaling pathway on expression of microRNAs, one new group of players important to carcinogenesis. Restoration of APC function in colorectal cancer cells led to the deregulation of several cancer-related microRNAs, such as miR-122a which was recognized as the liver-specific microRNA. MiR-122a was down-regulated in gastrointestinal cancer cell lines as well as primary carcinoma tissues. Inhibition of miR-122a could reverse wild-type APC-induced growth inhibition of gastrointestinal cancer cells while miR-122a mimic inhibited cell growth. In summary, we identified some cancer-related microRNAs regulated by APC/β-catenin signaling pathway. The down-regulation of miR-122a mediated by aberrant APC/β-catenin signaling is important to the pathogenesis of gastrointestinal cancers.     

The tumor suppressor adenomatous polyposis coli controls the direction in which a cell extrudes from an epithelium

Despite high rates of cell death, epithelia maintain intact barriers by squeezing dying cells out using a process termed cell extrusion. Cells can extrude apically into the lumen or basally into the tissue the epithelium encases, depending on whether actin and myosin contract at the cell base or apex, respectively. We previously found that microtubules in cells surrounding a dying cell target p115 RhoGEF to the actin cortex to control where contraction occurs. However, what controls microtubule targeting to the cortex and whether the dying cell also controls the extrusion direction were unclear. Here we find that the tumor suppressor adenomatous polyposis coli (APC) controls microtubule targeting to the cell base to drive apical extrusion. Whereas wild-type cells preferentially extrude apically, cells lacking APC or expressing an oncogenic APC mutation extrude predominantly basally in cultured monolayers and zebrafish epidermis. Thus APC is essential for driving extrusion apically. Surprisingly, although APC controls microtubule reorientation and attachment to the actin cortex in cells surrounding the dying cell, it does so by controlling actin and microtubules within the dying cell. APC disruptions that are common in colon and breast cancer may promote basal extrusion of tumor cells, which could enable their exit and subsequent migration.     

Adenomatous polyposis coli protein regulates the cellular response to DNA replication stress

The adenomatous polyposis coli (APC) tumor suppressor traffics between nucleus and cytoplasm to perform distinct functions. Here we identify a specific role for APC in the DNA replication stress response. The silencing of APC caused an accumulation of asynchronous cells in early S phase and delayed S phase progression in cells released from hydroxyurea-mediated replication arrest. Immunoprecipitation assays revealed a selective binding of APC to replication protein A 32kDa subunit (RPA32), and the APC-RPA32 complex increased at chromatin after hydroxyurea treatment. Interestingly, APC knock-down prevented accumulation at chromatin of the stress-induced S33- and S29-phosphorylated forms of RPA32, and reduced the expression of ATR-phosphorylated forms of S317-phospho-Chk1 and γ-H2AX. Using RPA32-inducible cells we showed that reconstitution of RPA32 diminished the S-phase delay caused by loss of APC. In contrast to full-length APC, the truncated APC mutant protein expressed in SW480 colon cancer cells was impaired in its binding and regulation of RPA32, and failed to regulate cell cycle after replication stress. We propose that APC associates with RPA at stalled DNA replication forks and promotes the ATR-dependent phosphorylation of RPA32, Chk1 and γ-H2AX in response to DNA replication stress, thereby influencing the rate of re-entry into the cell cycle.     

Adenomatous polyposis coli is down-regulated by the ubiquitin-proteasome pathway in a process facilitated by Axin

Adenomatous polyposis coli (APC) protein and Axin form a complex that mediates the down-regulation of beta-catenin, a key effector of Wnt signaling. Truncation mutations in APC are responsible for familial and sporadic colorectal tumors due to failure in the down-regulation of beta-catenin. While the regulation of beta-catenin by APC has been extensively studied, the regulation of APC itself has received little attention. Here we show that the level of APC is down-regulated by the ubiquitin-proteasome pathway and that Wnt signaling inhibits the process. The domain responsible for the down-regulation and direct ubiquitination was identified. We also show an unexpected role for Axin in facilitating the ubiquitination-proteasome-mediated down-regulation of APC through the oligomerization of Axin. Our results suggest a new mechanism for the regulation of APC by Axin and Wnt signaling.     

The Adenomatous polyposis coli tumour suppressor is essential for Axin complex assembly and function and opposes Axin's interaction with Dishevelled

Most cases of colorectal cancer are linked to mutational inactivation of the Adenomatous polyposis coli (APC) tumour suppressor. APC downregulates Wnt signalling by enabling Axin to promote the degradation of the Wnt signalling effector β-catenin (Armadillo in flies). This depends on Axin's DIX domain whose polymerization allows it to form dynamic protein assemblies ('degradasomes'). Axin is inactivated upon Wnt signalling, by heteropolymerization with the DIX domain of Dishevelled, which recruits it into membrane-associated 'signalosomes'. How APC promotes Axin's function is unclear, especially as it has been reported that APC's function can be bypassed by overexpression of Axin. Examining apc null mutant Drosophila tissues, we discovered that APC is required for Axin degradasome assembly, itself essential for Armadillo downregulation. Degradasome assembly is also attenuated in APC mutant cancer cells. Notably, Axin becomes prone to Dishevelled-dependent plasma membrane recruitment in the absence of APC, indicating a crucial role of APC in opposing the interaction of Axin with Dishevelled. Indeed, co-expression experiments reveal that APC displaces Dishevelled from Axin assemblies, promoting degradasome over signalosome formation in the absence of Wnts. APC thus empowers Axin to function in two ways-by enabling its DIX-dependent self-assembly, and by opposing its DIX-dependent copolymerization with Dishevelled and consequent inactivation.     

Adenomatous polyposis coli localization is both cell type and cell context dependent

The adenomatous polyposis coli (APC) tumor suppressor protein is mutated in most colorectal carcinomas. In addition to its role in WNT signaling it is proposed to be involved in both cell migration and mitosis. Although a variety of studies have shown an APC localization along lateral membranes of adjacent epithelial cells the existence of a cortical APC localization in mammalian cells remains controversial. To address this we have used matched rat epithelial (NRK-52E) and fibroblast (NRK-49F) cell lines to investigate the localization of APC. Subconfluent cultures of NRK-52E and -49F cells displayed microtubule-associated APC populations by immunostaining. However, confluent NRK-52E, but not -49F monolayers, exhibited a cortical APC distribution. Cortical APC localized in close proximity to a number of cell junction proteins in a microtubule-independent manner while calcium switch experiments suggested that APC was recruited to the cortex only when junction assembly was complete. Confluent NRK-49F and -52E cells also showed contrasting APC localizations in response to monolayer wounding. Our data suggests APC cortical localization is a feature of confluent epithelioid cells and that the subcellular distribution of APC is therefore dependent upon both cell type and context.     

Adenomatous polyposis coli regulates axon arborization and cytoskeleton organization via its N-terminus

Conditional deletion of APC leads to marked disruption of cortical development and to excessive axonal branching of cortical neurons. However, little is known about the cell biological basis of this neuronal morphological regulation. Here we show that APC deficient cortical neuronal growth cones exhibit marked disruption of both microtubule and actin cytoskeleton. Functional analysis of the different APC domains revealed that axonal branches do not result from stabilized β-catenin, and that the C-terminus of APC containing microtubule regulatory domains only partially rescues the branching phenotype. Surprisingly, the N-terminus of APC containing the oligomerization domain and the armadillo repeats completely rescues the branching and cytoskeletal abnormalities. Our data indicate that APC is required for appropriate axon morphological development and that the N-terminus of APC is important for regulation of the neuronal cytoskeleton.     

Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1

The tumor suppressor protein adenomatous polyposis coli (APC) regulates cell protrusion and cell migration, processes that require the coordinated regulation of actin and microtubule dynamics. APC localizes in vivo to microtubule plus ends and actin-rich cortical protrusions, and has well-documented direct effects on microtubule dynamics. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal “basic” domain of APC (APC-B) potently nucleates the formation of actin filaments in vitro and stimulates actin assembly in cells. Nucleation is achieved by a mechanism involving APC-B dimerization and recruitment of multiple actin monomers. Further, APC-B nucleation activity is synergistic with its in vivo binding partner, the formin mDia1. Together, APC-B and mDia1 overcome a dual cellular barrier to actin assembly imposed by profilin and capping protein. These observations define a new function for APC and support an emerging view of collaboration between distinct actin assembly–promoting factors with complementary activities.     

Adenomatous polyposis coli influences micronuclei induction by PhIP and acrylamide in mouse erythrocytes

Micronucleus (MN) induction in erythrocytes of multiple intestinal neoplasia (Min) mice with heterozygous Apc mutation was measured after s.c. injections of acrylamide, glycidamide, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and colchicine, and compared with wild-type (wt) mice. Since Apc influences microtubule dynamics, we wanted to test whether Min-mice were more sensitive to the production of MN than wild-type mice. We also examined the effect of pre-treatment with cytosine beta-D-arabinofuranoside (Ara C) and hydroxyurea, which inhibit ligation of DNA strand breaks in the repair of DNA adducts. All compounds induced a significant increase in MN in both strains of mice with the following potencies: acrylamide相似文献   

Identification of a link between the SAMP repeats of adenomatous polyposis coli tumor suppressor and the Src homology 3 domain of DDEF

The adenomatous polyposis coli (APC) tumor suppressor protein is a multifunctional protein with a well characterized role in the Wnt signal transduction pathway and in cytoskeletal regulation. The SAMP repeats region of APC, an Axin-binding site, is known to be important for tumor suppression and for the developmental function of APC. We performed a yeast two-hybrid screening using the first SAMP motif-containing region of Xenopus APC as bait and obtained several SAMP binding candidates including DDEF2 (development and differentiation enhancing factor 2), which is an ADP-ribosylation factor (Arf) GTPase-activating protein (GAP (ArfGAP)) involved in the regulation of focal adhesions. In vitro and in cells the Src homology 3 (SH3) domain of DDEF2 and its close homolog, DDEF1, are associated with the SAMP motif of APC competitively with Axin1. Moreover, NMR chemical shift perturbation experiments revealed that the SAMP motif interacts at the same surface of the SH3 domain of DDEF as the known SH3 binding motif, PXXP. When fluorescent protein-tagged APC and DDEF are expressed in Xenopus A6 cells, co-localization at microtubule ends is observed. Overexpression and RNA interference experiments indicate that APC and DDEFs cooperatively regulate the distributions of microtubules and focal adhesions. Our findings reveal that the SAMP motif of APC specifically binds to the SH3 domains of DDEFs, providing new insights into the functions of APC in cell migration.     

Adenomatous polyposis coli (APC) is essential for maintaining the integrity of the seminiferous epithelium

Sertoli cells provide the microenvironment necessary for germ cell development and spermatogenesis; disruption of Sertoli cell morphology or function can lead to germ cell aplasia, which is observed in testicular dysgenesis syndrome. Mutation of the adenomatous polyposis coli (APC) gene has been associated with various human cancers, including testicular cancer, but its involvement in nonmalignant testicular pathologies has not been reported. We have developed a mouse model (APC(cko)) that expresses a truncated form of APC in Sertoli cells. Despite normal embryonic and early postnatal testicular development in APC(cko) mice, premature germ cell loss and Sertoli cell-only seminiferous tubules were observed in mutant testes without affecting Sertoli cell quiescence, apoptosis, or differentiation, which were confirmed by the absence of both proliferating cell nuclear antigen, DNA strand breaks, and anti-Müllerian hormone, respectively. We show that mutant Sertoli cells lose their apical extensions, which would normally enclose germ cells during various stages of spermatogenesis, and were unable to maintain the blood-testis barrier because of disrupted expression of junctional proteins. We also observed an up-regulation of Snail and Slug, markers suggestive of epithelial-mesenchymal transition in the Sertoli cells, but tumorigenesis was not observed. No comparable phenotype was observed with Sertoli cell-specific loss-of-function mutations in β-catenin, leading us to speculate that truncation of APC in Sertoli cells results in progressive degeneration of the seminiferous tubules by a mechanism that disrupts the integrity of Sertoli cell junctions independently of APC-regulated β-catenin activities and leads to development of a Sertoli cell-only phenotype.