Gene name - APC-like
Synonyms - D-APC
Cytological map position - 98F1--98F14
Function - intracellular signaling protein
Symbol - Apc
Genetic map position - 3-
Classification - adenomatous polyposis coli homolog
Cellular location - cytoplasmic
|Recent literature||Suijkerbuijk, S. J., Kolahgar, G., Kucinski, I. and Piddini, E. (2016). Cell competition drives the growth of intestinal adenomas in Drosophila. Curr Biol [Epub ahead of print]. PubMed ID: 26853366
Tumor-host interactions play an increasingly recognized role in modulating tumor growth. Thus, understanding the nature and impact of this complex bidirectional communication is key to identifying successful anti-cancer strategies. It has been proposed that tumor cells compete with and kill neighboring host tissue to clear space that they can expand into; however, this has not been demonstrated experimentally. This study used the adult fly intestine to investigate the existence and characterize the role of competitive tumor-host interactions. This study shows that APC-/--driven intestinal adenomas compete with and kill surrounding cells, causing host tissue attrition. Importantly, this study demonstrates that preventing cell competition, by expressing apoptosis inhibitors, restores host tissue growth and contains adenoma expansion, indicating that cell competition is essential for tumor growth. It was further shown that JNK signaling is activated inside the tumor and in nearby tissue and is required for both tumor growth and cell competition. Lastly, it was found that APC-/- cells display higher Yorkie (YAP) activity than host cells and that this promotes tumor growth, in part via cell competition. Crucially, it was found that relative, rather than absolute, Hippo activity determines adenoma growth. Overall, these data indicate that the intrinsic over-proliferative capacity of APC-/- cells is not uncontrolled and can be constrained by host tissues if cell competition is inhibited, suggesting novel possible therapeutic approaches.
|Wang, Z., Tacchelly-Benites, O., Yang, E. and
Ahmed, Y. (2016). Dual roles
for membrane association of Drosophila Axin in Wnt signaling.
PLoS Genet 12: e1006494. PubMed ID: 27959917
Axin, a concentration-limiting scaffold protein, facilitates assembly of a "destruction complex" that prevents Wnt signaling in the unstimulated state and a plasma membrane-associated "signalosome" that activates signaling following Wnt stimulation. In the classical model, Axin is cytoplasmic under basal conditions, but relocates to the cell membrane after Wnt exposure. This study analyzed the subcellular distribution of endogenous Drosophila Axin in vivo and found that a pool of Axin localizes to cell membrane proximal puncta even in the absence of Wnt stimulation. Axin localization in these puncta is dependent on the destruction complex component Adenomatous polyposis coli (Apc). In the unstimulated state, the membrane association of Axin increases its Tankyrase-dependent ADP-ribosylation and consequent proteasomal degradation to control its basal levels. Furthermore, Wnt stimulation does not result in a bulk redistribution of Axin from cytoplasmic to membrane pools, but causes an initial increase of Axin in both of these pools, with concomitant changes in two post-translational modifications, followed by Axin proteolysis hours later. Finally, the ADP-ribosylated Axin that increases rapidly following Wnt stimulation is membrane associated. The study concludes that even in the unstimulated state, a pool of Axin forms membrane-proximal puncta that are dependent on Apc, and that membrane association regulates both Axin levels and Axin's role in the rapid activation of signaling that follows Wnt exposure.
|Tian, A., Benchabane, H., Wang, Z., Zimmerman, C., Xin, N., Perochon, J., Kalna, G., Sansom, O. J., Cheng, C., Cordero, J. B. and Ahmed, Y. (2017). Intestinal stem cell overproliferation resulting from inactivation of the APC tumor suppressor requires the transcription cofactors Earthbound and Erect wing. PLoS Genet 13(7): e1006870. PubMed ID: 28708826
Wnt/beta-catenin signal transduction directs intestinal stem cell (ISC) proliferation during homeostasis. Hyperactivation of Wnt signaling initiates colorectal cancer, which most frequently results from truncation of the tumor suppressor Adenomatous polyposis coli (APC). Using a Drosophila model this study demonstrated that two evolutionarily conserved transcription cofactors, Earthbound (Ebd) and Erect wing (Ewg), are essential for all major consequences of Apc1 inactivation in the intestine: the hyperactivation of Wnt target gene expression, excess number of ISCs, and hyperplasia of the epithelium. In contrast, only Ebd, but not Ewg, mediates the Wnt-dependent regulation of ISC proliferation during homeostasis. Therefore, in the adult intestine, Ebd acts independently of Ewg in physiological Wnt signaling, but cooperates with Ewg to induce the hyperactivation of Wnt target gene expression following Apc1 loss. These findings have relevance for human tumorigenesis, as Jerky (JRK/JH8), the human Ebd homolog, promotes Wnt pathway hyperactivation and is overexpressed in colorectal, breast, and ovarian cancers.
|Saito-Diaz, K., Benchabane, H., Tiwari, A., Tian, A., Li, B., Thompson, J. J., Hyde, A. S., Sawyer, L. M., Jodoin, J. N., Santos, E., Lee, L. A., Coffey, R. J., Beauchamp, R. D., Williams, C. S., Kenworthy, A. K., Robbins, D. J., Ahmed, Y. and Lee, E. (2018). APC inhibits ligand-independent Wnt signaling by the Clathrin endocytic pathway. Dev Cell 44(5): 566-581. PubMed ID: 29533772
Adenomatous polyposis coli (APC) mutations cause Wnt pathway activation in human cancers. Current models for APC action emphasize its role in promoting beta-catenin degradation downstream of Wnt receptors. Unexpectedly, this study found that blocking Wnt receptor activity in APC-deficient cells inhibits Wnt signaling independently of Wnt ligand. Inducible loss of APC is rapidly followed by Wnt receptor activation and increased beta-catenin levels. In contrast, APC2 loss does not promote receptor activation. This study shows that APC exists in a complex with clathrin and that Wnt pathway activation in APC-deficient cells requires clathrin-mediated endocytosis. Finally, conservation of this mechanism in was demonstrated in Drosophila intestinal stem cells. A model is proposed in which APC and APC2 function to promote beta-catenin degradation, and APC also acts as a molecular "gatekeeper" to block receptor activation via the clathrin pathway.
|Popow, O., Paulo, J. A., Tatham, M. H., Volk, M. S., Rojas-Fernandez, A., Loyer, N., Newton, I. P., Januschke, J., Haigis, K. M. and Nathke, I. (2019). Identification of endogenous Adenomatous polyposis coli interaction partners and beta-catenin-independent targets by proteomics. Mol Cancer Res. PubMed ID: 31160382
Adenomatous Polyposis Coli (APC) is the most frequently mutated gene in colorectal cancer. APC negatively regulates the Wnt signaling pathway by promoting the degradation of beta-catenin, but the extent to which APC exerts Wnt/beta-catenin-independent tumor suppressive activity is unclear. To identify interaction partners and beta-catenin-independent targets of endogenous, full-length APC, label-free and multiplexed tandem mass tag-based mass spectrometry was applied. Affinity enrichment-mass spectrometry identified more than 150 previously unidentified APC interaction partners. Moreover, a global proteomic analysis revealed that roughly half of the protein expression changes that occur in response to APC loss are independent of beta-catenin. Combining these two analyses, Misshapen-like kinase 1 (MINK1) was identifed as a putative substrate of an APC-containing destruction complex. The interaction was validated between endogenous MINK1 and APC and further confirmed the negative - and beta-catenin-independent -regulation of MINK1 by APC. Increased Mink1/Msn levels were also observed in mouse intestinal tissue and Drosophila follicular cells expressing mutant Apc/APC when compared to wild-type tissue/cells. Collectively, the results highlight the extent and importance of Wnt-independent APC functions in epithelial biology and disease. The tumor suppressive function of APC - the most frequently mutated gene in colorectal cancer - is mainly attributed to its role in beta-catenin/Wnt signaling. This study substantially expands the list of APC interaction partners and reveals that approximately half of the changes in the cellular proteome induced by loss of APC function are mediated by beta-catenin-independent mechanisms.
|Bensard, C. L., Wisidagama, D. R., Olson, K. A., Berg, J. A., Krah, N. M., Schell, J. C., Nowinski, S. M., Fogarty, S., Bott, A. J., Wei, P., Dove, K. K., Tanner, J. M., Panic, V., Cluntun, A., Lettlova, S., Earl, C. S., Namnath, D. F., Vazquez-Arreguin, K., Villanueva, C. J., Tantin, D., Murtaugh, L. C., Evason, K. J., Ducker, G. S., Thummel, C. S. and Rutter, J. (2019). Regulation of tumor initiation by the mitochondrial pyruvate carrier. Cell Metab. PubMed ID: 31813825
Although metabolic adaptations have been demonstrated to be essential for tumor cell proliferation, the metabolic underpinnings of tumor initiation are poorly understood. This study found that the earliest stages of colorectal cancer (CRC) initiation are marked by a glycolytic metabolic signature, including downregulation of the mitochondrial pyruvate carrier (MPC), which couples glycolysis and glucose oxidation through mitochondrial pyruvate import. Genetic studies in Drosophila suggest that this downregulation is required because hyperplasia caused by loss of the Apc or Notch tumor suppressors in intestinal stem cells can be completely blocked by MPC overexpression. Moreover, in two distinct CRC mouse models, loss of Mpc1 prior to a tumorigenic stimulus doubled the frequency of adenoma formation and produced higher grade tumors. MPC loss was associated with a glycolytic metabolic phenotype and increased expression of stem cell markers. These data suggest that changes in cellular pyruvate metabolism are necessary and sufficient to promote cancer initiation.
|Ngo, S., Liang, J., Su, Y. H. and O'Brien, L. E. (2020). Disruption of EGF Feedback by Intestinal Tumors and Neighboring Cells in Drosophila. Curr Biol 30(8): 1537-1546. PubMed ID: 32243854
In healthy adult organs, robust feedback mechanisms control cell turnover to enforce homeostatic equilibrium between cell division and death. Nascent tumors must subvert these mechanisms to achieve cancerous overgrowth. Elucidating the nature of this subversion can reveal how cancers become established and may suggest strategies to prevent tumor progression. In adult Drosophila intestine, a well-studied model of homeostatic cell turnover, the linchpin of cell equilibrium is feedback control of the epidermal growth factor (EGF) protease Rhomboid (Rho). Expression of Rho in apoptotic cells enables them to secrete EGFs, which stimulate nearby stem cells to undergo replacement divisions. As in mammals, loss of adenomatous polyposis coli (APC) causes Drosophila intestinal stem cells to form adenomas. This study demonstrates that Drosophila APC(-/-) tumors trigger widespread Rho expression in non-apoptotic cells, resulting in chronic EGF signaling. Initially, nascent APC(-/-) tumors induce rho in neighboring wild-type cells via acute, non-autonomous activation of Jun N-terminal kinase (JNK). During later growth and multilayering, APC(-/-) tumors induce rho in tumor cells by autonomous downregulation of E-cadherin (E-cad) and consequent activity of p120-catenin. This sequential dysregulation of tumor non-autonomous and -autonomous EGF signaling converts tissue-level feedback into feed-forward activation that drives cancerous overgrowth. Because Rho, EGF receptor (EGFR), and E-cad are associated with colorectal cancer in humans [10-17], these findings may shed light on how human colorectal tumors progress.
Because adenomatous polyposis coli (APC) was first identified in vertebrates, this review will initially focus on the vertebrate protein before dealing with specific information about the Drosophila homolog, termed APC-like, or simply, Apc. APC, a gene mutated in familial adenomatous polyposis and sporadic colorectal tumors, is a tumor suppressor and a component of the WNT pathway (see Drosophila Wingless) functioning as a negative regulator in signal transduction. The vertebrate APC protein binds to ß-catenin (the vertebrate homolog of Armadillo) and accelerates ß-catenin turnover. APC also binds to Zeste white-3 (the vertebrate homolog of Drosophila Shaggy) and is in fact a better substrate for Zw-3 than ß-catenin. A complex between these three proteins may keep ß-catenin levels low (Rubinfeld, 1996). Indeed, APC, Zeste white-3 and Axin are part of a multi-molecular complex termed "ß-catenin destruction complex." APC, E-cadherin, and TCF (the vertebrate homolog of Pangolin) can all bind to the middle domain of ß-catenin, the part that contains a stretch of 13-fold repeated sequences, but each partner binds to a specific subset of these repeats. Possibly, there is some competition for binding, and binding by one or the other partners is subject to some regulation (Nusse, 1997).
APC traces its history to tumors found in familial colon carcinomas; it is a tumor suppressor gene that is deleted in tumors or in germ line DNA. The findings on interactions between Arm and Pangolin in Drosophila have also led to a much better understanding of the mechanism of tumorigenesis as a result of APC loss. Korinek (1997) has shown that several colon carcinoma lines, derived from tumors with APC deletions, contain a complex between one of the human TCF homologs (hTCF-4) and ß-catenin. This complex activates expression of reporter constructs, indicating that the loss of APC function in these cells releases enough ß-catenin to team up with TCF to become a transcriptional activator. As predicted from the model in which APC is a gatekeeper of ß-catenin, transfection of full-length APC into those cells inhibits expression of the reporter gene constructs. Mutant forms of APC, when transfected, are incapable of blocking target gene expression. These experiments provide a plausible explanation of how loss of APC can activate gene expression and lead to cell transformation (although no target genes in oncogenesis are known). A surprise came when Morin (1997) found several colon carcinoma cell lines with normal levels of wild-type APC but nevertheless displaying strong expression of these reporter constructs). It appeared that ß-catenin in those cells had found another way to escape from APC control: by mutations in its own gene. These mutations are also present in several melanomas (Robbins, 1996 and Rubinfeld, 1997 ) and form a special class: they eliminate specific amino acids in the amino-terminal domain of the protein, in particular residues that become phosphorylated prior to the proteolytic down-regulation of ß-catenin. Hence, ß-catenin can turn into an oncogene in its own right. Together, these findings suggest a model in which APC acts to regulate ß-catenin negatively. Inactivation of APC, the model suggests, results in elevation of ß-catenin levels and subsequent cellular transformation. However, not all data are consistent with this model (Nusse, 1997).
Keeping in mind the hypothesized role for APC in vertebrates as the gatekeeper for ß-catenin, it is of interest to explore the role on APC in Drosophila, especially because of the in depth understanding of the role of Armadillo (the Drosophila homolog of ß-catenin) and Pangolin in Wingless signaling, described briefly, in the two paragraphs below.
The roles of Armadillo (Arm) in both Wingless signal transduction and cellular adhesion have been extensively studied, making the fly a particularly amenable model system for the analysis of the regulation of ß-catenin by Apc. The cytoplasmic localization of Arm is regulated by phosphorylation. Shaggy/Zeste-white 3, a serine threonine kinase, lies upstream of Arm, and positively regulates the phosphorylation of Arm. Tyrosine phosphorylation modifies the adherens junction, increases the level of cytoplasmic rather than junctional Arm, but probably does not result in disassembly of the junction. Wingless signal negatively regulates Arm phosphorylation (Peifer, 1994).
How do Wingless signals get to the nucleus? The pathway has now been worked out in Drosophila, based on studies with vertebrates. A maternally expressed Xenopus homolog of mammalian HMG box factors (XTcf-3) binds to ß-catenin (the vertebrate homolog of Armadillo). XTcf-3 (Drosophila homolog: Pangolin) is a transcription factor that mediates ß-catenin-induced axis formation in Xenopus embryos. It has been proposed that the ß-catenin-XTcf-3 complex is responsible for activation of targets genes in response to upstream Wnt signals, which allow cytoplasmic ß-catenin to interact with XTcf-3 (Molenaar, 1996). These observations suggest that Armadillo, plus the associated transcription factor, Pangolin, are the nuclear effectors of Wingless.
With the cloning of Drosophila APC-like and the isolation of mutants, analysis of Drosophila Apc has begun in earnest. Drosophila Apc interacts in cell culture with vertebrate ßcatenin. Expression of the domain of Apc homologous to the region required for ß-catenin down-regulation results in down-regulation of intracellular ß-catenin in a mammalian cell line. This same region binds in vitro to the Armadillo protein. In spite of the demonstrated interaction of Drosophila Apc with Armadillo, no role for Apc could be demonstrated in Drosophila segmentation. APC mRNA and protein expression is very low, if detectable at all, during stages when Arm protein accumulates in a striped pattern in the epidermis of Drosophila embryos. Removing zygotic Apc expression does not alter Arm protein distribution, and the final cuticle pattern is not affected significantly (Hayashi, 1997). Of great interest is the finding of a second Drosophila Apc gene by the Berkeley Drosophila Genome Project (Ahmed, 1998). Perhaps this second Apc gene plays a role in segmentation.
Given the expectation of a role for Drosophila Apc in segmentation, the demonstration of a lack of Apc involvement in this process was truely a disappointment. Nevertheless, high levels of Apc expression have been detected in the central nervous system, suggesting a role for Apc in central nervous system development (Hayashi, 1997). A deficiency in the chromosomal region to which the Apc gene maps was used as the basis for genetic screens to isolate mutants in Apc. Potential mutants in Apc were identified by staining embryos from various mutant lines with a polyclonal antibody directed against the central portion of Apc. In wild-type embryos at early embryonic stages, Apc is detected in the pole cells, which ultimately give rise to the adult germline. This Apc expression is provided maternally and persists through the course of pole cell migration to the gonads. At later embryonic stages, strong zygotic Apc expression is found in both the CNS and peripheral nervous system, within the axon fiber tracts. Three mutants with a reduction in this zygotic expression were identified. Apc pole cell expression, provided by heterozygous mothers, persists in homozygous mutant embryos, thus demonstrating that Apc is provided maternally (Ahmed, 1998).
Phenotypic analysis of Drosophila Apc mutants has demonstrated a role for the gene in eye development. The normal compound eye structure consists of approximately 750 highly ordered eye units, termed ommatidia. Each ommatidium contains eight neuronal photoreceptor cells surrounded by retinal pigment cells. In contrast to the normal or wild-type animal, on eclosion the homozygous Apc mutant shows complete loss of all retinal neurons within all ommatidia, while the pigment cell lattice remains intact. Several days after eclosion, the pigment cells enlarge to the point of confluence (Ahmed, 1998).
Third instar larval eye imaginal discs (the precursors of the adult eye) from homozygous Apc mutants were analyzed prior to development of the photoreceptor phenotype, to determine whether photoreceptor cell loss is a consequence of defects in the initial formation of neurons or from defects in their subsequent differentiation. The mutant eye discs were examined for expression of Neurotactin, a neuronal-specific transmembrane protein that is used as a marker for photoreceptor cells. Both at the level of patterning of the ommatidial arrays and at the level of individual photoreceptor cells, the Neurotactin antigen staining is normal in the Apc mutant. As well, retinal axonal projections to the optic lobe are intact. This indicates that the initial photoreceptor cell formation proceeds normally in Apc mutants and that the defect seen in the mutant adult is the result of neuronal degeneration. Further studies have determined that the neuronal degeneration in the Apc mutant is a result of programmed cell death (apoptosis). Expression of p35, a baculoviral protein that interferes with apoptosis by inhibiting the function of caspase proteases, rescues the Apc mutant phenotype. Despite the dramatic rescue from death of Apc mutant retinal neurons by p35, there is a striking feature that distinguishes the rescued photoreceptors. In wild-type adult eyes, the retinal neurons extend the entire length of the ommatidia, tapering gradually in diameter from their apical to basal regions. In contrast, while the rescued Apc mutant photoreceptors display an intact morphology at the apical surface of the eye, at more basal levels, their diameters are dramatically shrunken, and they lose contact with their neighbors. This abnormal morphology is thought to reflect an arrest in differentiation and that this arrest accompanies the apoptotic death observed in the Apc mutant eye (Ahmed, 1998).
To test for Apc regulation of Arm activity genetically, Arm levels in the Apc mutant were lowered by replacing one wild-type copy of the arm gene with an null allele. When the wild-type arm gene dosage is reduced by one-half in the Apc mutant, many neurons survive. The overall efficiency of rescue achieved by halving the Arm dosage is similar to that obtained by ectopic p35 expression; however, one striking difference between the two is that the neurons rescued in the arm heterozygotes appear completely normal from apex to base. Thus, an inactivating arm mutation is a dominant suppressor of both the differentiation defect and the cell death of retinal neurons in the Apc mutant. These findings provide genetic evidence that Apc functions to regulate Arm negatively and suggest that in the absence of Apc, an increase in Arm activity results in both a differentiation defect and apoptotic cell death (Ahmed, 1998).
To test this idea, the effects of elevated levels of Arm on photoreceptor differentiation were analyzed using the UAS/GAL4 system to overexpress Arm in retinal neurons. Neuronal-specific overexpression of Arm under control of the elav-GAL4 transactivator results in photoreceptor loss that is phenotypically similar to, but weaker than, that seen in Apc mutants, with some ommatidia losing all photoreceptor cells, and most others having a reduction in their number. This reveals that death of photoreceptors is sensitive to Arm dosage and suggests the requirement for titration of Apc activity. To examine further the effects of Arm levels on photoreceptor death, mutations in the amino terminus of Arm were used that reduce the rate at which it is degraded. Neuronal-specific overexpression of an amino-terminal deletion of Arm that results in its stabilization, directed by the elav-GAL4 transactivator, results in the loss of all neurons in all ommatidia, and only pigment cells remain. Overexpression of stabilized Arm under control of the sevenless-GAL4 transactivator, which directs strong expression in 3 of the 8 photoreceptor cells, and weak expression in 2 others, results in photoreceptor loss of only a fraction of cells per ommatidium. Together, these findings demonstrate that overexpression of Arm within retinal cells committed to a neuronal fate results in their death and suggest that the Apc mutant phenotype is mediated by elevation of Arm activity (Ahmed, 1998).
To characterize Arm's ability to activate cell death, and further examine the role of Apc in the Wingless pathway, effects brought about by other members of the Wingless signaling pathway on the apoptosis that is induced by Apc loss were also examined. One well-characterized negative regulator of Arm's signal transduction function is the serine/threonine kinase Zeste-white 3 (Zw3). Inactivation of Zw3 yields elevated levels of cytoplasmic Arm but has little effect on Arm's function in junctional complexes. Neuronal-specific overexpression of Zw3, directed by the elav-GAL4 transactivator, rescues many retinal neurons from apoptosis in the Apc mutant. Remarkably, the rescued cells are detected solely at the apical surface of the eye; more basal sections reveal no rescue. Thus, although the underlying differentiation defect persists, overexpression of Zw3 prevents retinal cell death in the Apc mutant, suggesting a role for cytoplasmic Arm in the activation of apoptosis (Ahmed, 1998).
The signaling mediated by Arm requires not only its cytoplasmic accumulation but also the activity of the DNA-binding protein Drosophila TCF known as Pangolin (Pan). Arm directly interacts with Pangolin through its central Armadillo repeats, a region that is critical in Arm's ability to induce cell death. Pangolin function in an Apc mutant background cannot be completely eliminated; however, flies heterozygous for two mutant alleles of pangolin, panciD and pan13, were examined to determine if Pangolin is required in the activation of cell death that results from Apc loss. panciD is mutant for two loci, ci and pan; it behaves as both a hypomorphic allele of ci and a null allele of pan. In flies heterozygous for panciD, some retinal neurons are rescued from cell death in the Apc mutant. As was seen with ectopic Zw3 expression, the rescued cells are detected solely at the apical surface of the eye. The same rescue is found in Apc mutants that are heterozygous for the null allele pan13. These findings suggest that elevated Arm levels activate a cell death pathway via the Arm/Pangolin complex (Ahmed, 1998).
Apc mutations in Drosophila reveal a surprising phenotype that parallels the retinal defects induced by APC loss in humans. In both species, retinal lesions composed of degenerated photoreceptor neurons and enlarged pigment cells are formed during eye development, suggesting a particular sensitivity of these cell types to APC loss. There has been some debate as to whether the degeneration of retinal neurons found in congenital hypertrophy of the retinal pigment epithelium (CHRPE) is a secondary consequence of a functional defect in the pigment cells or vice versa. Both the cell death that results from overexpression of Arm specifically within neurons, as well as the curious expression pattern of Arm and Apc in retinal neurons, suggest that in the fly, retinal degeneration in the setting of APC loss is the result of a defect within the photoreceptor cells themselves (Ahmed, 1998 and references).
Is there a role for Armadillo and Pangolin in the induction of retinal cell death during the normal development of the Drosophila eye? Apoptosis occurs within every developing ommatidium to eliminate two to three extraneous cells, a requirement in the refining of the highly ordered compound eye structure. Disruption of this process results in the survival of additional pigment cells in each ommatidium, and a disordering of the retina. Despite the extensive apoptosis that occurs during retinal development, there is little cell death in photoreceptors, and there is no evidence supporting a role for Arm in normally occurring retinal cell death. Rather, the results of the current study suggest that the normal differentiation of these neurons requires tight regulation of Arm activity. In the absence of this regulation, a differentiation defect occurs. A model is favored in which Pangolin, via the Arm/Pangolin complex, acts as a sensor of unregulated elevation of Arm; in response to this elevation, a cell death pathway is triggered. This Pangolin sensing mechanism would provide an Arm-based molecular link between a gross aberration in neuronal differentiation and the concomitant activation of cell death (Ahmed, 1998).
Mammalian ß-catenin is proteolytically processed in apoptotic cells, in a manner that appears caspase dependent. As the cleavage of ß-catenin renders it unable to function in adherens junctions, and the disruption of such junctions is an early event in programmed cell death, this processed form has been proposed to be an effector of apoptosis (Brancolini, 1997). In contrast, the data for Drosophila suggest that at least in retinal neurons, ß-catenin could function not only as an effector but also as an activator of the cell death machinery. The processing of ß-catenin in apoptotic cells generates a deletion of the amino terminus that is predicted to result in its stabilization (Brancolini, 1997). If caspase-mediated proteolytic processing of Arm occurs in apoptotic photoreceptor cells, it might generate a positive feedback loop, whereby elevated levels of Arm would activate an apoptotic pathway; this induction of apoptosis would result in the cleavage and stabilization of Arm, which in turn would reinforce the activation of the apoptotic pathway. APC itself is specifically proteolytically processed to generate a 90 kDa amino-terminal fragment in colon carcinoma cells undergoing apoptotic death (Browne, 1994). As such, cleavage would likely eliminate APC's ability to regulate ß-catenin negatively, this would further reinforce a positive feedback loop, increasing ß-catenin levels in apoptotic cells. In this regard, the markedly increased levels of apoptosis found in the colonic polyps, but not in the surrounding mucosa, from humans with germline mutations in APC are intriguing (Sträter, 1995). Together, these findings from varied experimental systems implicate ß-catenin and APC as important regulators not only of cell differentiation but also of programmed cell death (Ahmed, 1998).
The mechanisms by which the Wingless (Wg) morphogen modulates the activity of the transcriptional activator Armadillo (Arm) to elicit precise, concentration-dependent cellular responses remain uncertain. Arm is targeted for proteolysis by the Axin/Adenomatous polyposis coli (Apc1 and Apc2)/Zeste-white 3 destruction complex, and Wg-dependent inactivation of destruction complex activity is crucial to trigger Arm signaling. In the prevailing model for Wg transduction, only Axin levels limit destruction complex activity, whereas Apc is present in vast excess. To test this model, Apc activity was reduced to different degrees, and the effects were analyzed on three concentration-dependent responses to Arm signaling that specify distinct retinal photoreceptor fates. It was found that both Apc1 and Apc2 negatively regulate Arm activity in photoreceptors, but that the relative contribution of Apc1 is much greater than that of Apc2. Unexpectedly, a less than twofold reduction in total Apc activity, achieved by loss of Apc2, decreases the effective threshold at which Wg elicits a cellular response, thereby resulting in ectopic responses that are spatially restricted to regions with low Wg concentration. It is concluded that Apc activity is not present in vast excess, but instead is near the minimal level required for accurate graded responses to the Wg morphogen (Benchabane, 2008).
Previous genetic studies have provided conclusive evidence that the two Drosophila Apc proteins are crucial negative regulators of Arm signaling. Simultaneous inactivation of both Apc proteins results in ectopic Arm signaling in nearly all, if not all, cells, indicating that Apc is required to prevent Arm signaling in the absence of Wg stimulation. In contrast with the prevailing model for Wg transduction, which proposes that Apc is present in vast excess, the work presented in this study reveals that a less than twofold reduction in Apc activity can shift the threshold for the response to Wg. It is concluded that by negatively regulating Arm, Apc prevents ectopic Arm activity not only where Wg is absent, but also within the range of the Wg gradient (Benchabane, 2008).
Translation of a gradient of Wg morphogen activity to quantitatively distinct levels of Arm signaling is required to induce concentration-dependent cellular responses, although the mechanisms by which this occurs remain uncertain. The current results reveal that in regions of low Wg concentration, reducing total Apc activity by less than twofold results in aberrant cell fate specification. A morphogen model predicts that the low Wg concentration present in this region of the gradient is below the threshold necessary to trigger a detectable cellular response. This is the only region within the Wg gradient where a relatively small reduction in total Apc activity elicits an ectopic cellular response, and this response is characteristic of intermediate-level Arm signaling. Thus, these results reveal that Apc activity is in excess in regions where Wg is absent, but is not in vast excess within the range of the Wg gradient. Together, these data indicate that Apc activity is present near the minimal level required to prevent ectopic Arm signaling and thereby ensure accurate graded responses (Benchabane, 2008).
In Xenopus egg extracts, the levels of Axin are several magnitudes lower than the levels of other proteins in the destruction complex, suggesting that only Axin is a limiting component in Arm proteolysis, whereas Apc is present in vast excess. How can these biochemical data be reconciled with the current in vivo data, which indicate that Apc is not present in excess within the range of the Wg gradient? One possibility is that the levels of Apc in Xenopus eggs are much greater than those present in Drosophila photoreceptors. Alternatively, total Apc levels could be present in excess regardless of cell type or organism, but the relevant pool contributing to destruction complex activity, distinguished by either post-translational modification and/or intracellular localization, might be present near threshold levels. A correlation between the degree of reduction in the activity of the fly and mammalian Apc proteins with the level of β-catenin/Arm signaling has been demonstrated in several other developmental contexts and in tumorigenesis. Thus data from diverse experimental models indicate that the level of Apc contributes to the level of β-catenin/Arm signaling (Benchabane, 2008).
How is a gradient of Wg concentration translated into quantitatively distinct levels of Arm activity? Upon Wg stimulation, inactivation of the Axin/Zw3/Apc destruction complex is the primary event that triggers Arm signaling. Inactivation of Axin is important for downstream signal transduction in response to Wg stimulation, and is likely to be mediated by the translocation of Axin to the plasma membrane, and/or the degradation of Axin. Thus the local Axin concentration is likely to have a significant role in determining whether the destruction complex is assembled, and consequently is important in regulating Arm stability. The current findings provide in vivo evidence that the level of destruction complex activity is crucial for accurate patterning in response to Wg, and is dependent not only on Axin, but also on the maintenance of Apc activity above a minimal level. It is concluded that within the range of the Wg gradient, both Axin and Apc are present near threshold levels, and that, together, they achieve the precise levels of destruction complex activity required for accurate graded responses (Benchabane, 2008).
In many differentiated cells, microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control these arrays have been identified. For example, mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. Drosophila neurons contain uniform-polarity minus-end-out microtubules in dendrites, which are often highly branched. Because undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity, this study used this system to understand how cells can maintain dynamic arrays of polarized microtubules. Growing microtubules navigate dendrite branch points by turning the same way, toward the cell body, 98% of the time, and growing microtubules track along stable microtubules toward their plus ends. Using RNAi and genetic approaches, this study shows that kinesin-2, and the +TIPS EB1 and APC, are required for uniform dendrite microtubule polarity. Moreover, the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to branch points suggests that these proteins work together at dendrite branches. The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. It is concluded that microtubule growth is directed at dendrite branch points and that kinesin-2, APC, and EB1 are likely to play a role in this process. It is propose that Kinesin-2 is recruited to growing microtubules by +TIPS and that the motor protein steers growing microtubules at branch points. This represents a newly discovered mechanism for maintaining polarized arrays of microtubules (Mattie, 2010).
Most cells in multicellular organisms contain polarized noncentrosomal microtubule arrays. In interphase mammalian cultured cells, microtubules are nucleated at the centrosomal microtubule organizing center (MTOC), and plus ends grow towards the cell periphery. However, in many differentiated cells, minus ends are not focused at a centrosomal MTOC. In epithelial cells, a major population of microtubules has minus ends focused at the apical side and plus ends at the basal side. In muscle cells, minus ends spread out around the nuclear envelope, and neurons have perhaps the simplest and most strikingly polarized noncentrosomal microtubule arrays. The mechanisms that organize these noncentrosomal microtubule arrays are poorly understood (Mattie, 2010).
Neurons have two types of processes that extend from the cell body: axons and dendrites. Dendrites primarily receive signals from other neurons or the environment, and axons send signals to other neurons or output cells. One basic difference between axons and dendrites is the arrangement of microtubules. In axons microtubules are arranged into an overlapping array of uniform polarity plus-end-out microtubules. In dendrites of cultured mammalian neurons microtubules have mixed orientation near the cell body. In dendrites of Drosophila neurons 90%-95% of microtubules have minus ends distal to the cell body. As the dendritic array in Drosophila is very simple, and extremely different from a centrosomal array, This study used it as a model system to identify mechanisms that organize polarized noncentrosomal microtubules (Mattie, 2010).
It is not known how uniform dendrite microtubule polarity is established or maintained. Models for generating the plus-end-out axonal microtubule array focus on sliding of microtubules by motor proteins. In mammalian neurons, microtubules are thought to be nucleated in the cell body at the centrosome, then released from the centrosome and transported down the axon in the correct orientation by motors including dynein. Models to account for mixed orientation microtubules in dendrites of cultured neurons have also been proposed. In this case, the kinesin MKLP1 (Kif23) has been proposed to transport minus-end-out microtubules into dendrites along plus-end-out microtubules. The current study identified a new mechanism that is required for uniform microtubule polarity in dendrites (see Interactions between kinesin-2 and +TIPs, and localization of Apc2-GFP to dendrite branch points). As it uses conserved, generally expressed proteins, it could play a role in maintaining microtubule polarity in many other cell types (Mattie, 2010).
Using Drosophila dendrites as a model system, this study demonstrates that growing microtubule plus ends almost always turn towards the cell body at branch points, and that they track stable microtubules through branches. Kinesin-2, EB1 and APC are all required to maintain microtubule polarity and are linked in an interaction network. Based on these results, a model is proposed for directed growth of microtubules in dendrites. Apc2 most likely contains a branch localization signal because Apc2-GFP localizes well to dendrite branches even when overexpressed. Localization of Apc2 to dendrite branch points can recruit Apc to the branch. Apc can interact with the Kap3 subunit of kinesin-2, and so could increase concentration of the motor near the branch. EB1-GFP does not concentrate at branches, so it is proposed that a growing microtubule plus end coated with EB1 is transiently linked to kinesin-2 as it passes through the branch, through the interaction between Apc and the EB1 tail. SxIP motifs in Apc and Klp68D could also contribute to this interaction. As both Kap3 and the SxIP motif in Klp68D are in the kinesin-2 tail, the motor domain would be free to walk along a nearby stable microtubule towards the plus end and cell body (Mattie, 2010).
Even a very brief application of force pulling the growing microtubule towards the cell body should be sufficient to steer growth towards the cell body. Once the tip of the microtubule turns, growth would be constrained by the dendrite walls. The association of the growing plus end with stable microtubule would probably only need to be maintained over a distance of a micron. This model is consistent with the observations that kinesin-2 has shorter run lengths than kinesin-1, and that individual EB1 interactions with the microtubule plus end persist less than a second (Mattie, 2010).
Observations of plus end behavior in vivo also favor a model in which only transient interactions of the motor and microtubule plus end are involved, because plus ends turning sharply are frequently seen. Stable microtubules do not accommodate such sharp turns, so the sharp turns of plus ends are not likely to occur while the plus end is tracking along a stable microtubule. Instead they likely represent a switch from a freely growing plus end to one that is following a track (Mattie, 2010).
Directed growth of microtubules at dendrite branch points allows dendrites to maintain uniform minus-end-out polarity despite continued microtubule remodeling. This mechanism is also likely necessary to establish uniform microtubule polarity in branched dendrites, but probably cannot account for minus-end-out polarity on its own. It is hypothesized that directed microtubule growth is used in concert with an unidentified mechanism to control microtubule polarity in dendrites. Transport of oriented microtubule pieces has been proposed to contribute to axon microtubule polarity and a similar mechanism could play a role in dendrites. For example, a kinesin anchored to the cell cortex by its C-terminus could shuttle minus-end-out microtubule seeds into dendrites. Alternately, polarized microtubule nucleation sites could be localized within dendrites. Identifying directed growth as one mechanism that contributes to dendrite microtubule polarity should facilitate identification of the missing pieces of this puzzle (Mattie, 2010).
Because kinesin-2, APC, EB1 and polarized microtubules are found in many cell types, directed growth of microtubules along stable microtubule tracks could be very broadly used for maintaining microtubule polarity. This is a newly identified function for both the +TIP proteins and kinesin-2. APC has previously been localized to junctions between a microtubule tip and the side of another microtubule at the cortex of epithelial cells, and sliding of microtubule tips along the side of microtubules was also seen in this system, but there was no association with a particular direction of movement or overall microtubule polarity (Mattie, 2010).
As kinesin-2 has previously been shown to be enriched in tips of growing axons in cultured mammalian neurons, as has APC, it is possible that they could mediate tracking of growing microtubules along existing microtubule tracks in the growth cone. In fact, this type of microtubule tracking behavior has been observed in axonal growth cones under low actin conditions. Thus directed growth of microtubules could also be important during axon outgrowth. Indeed, the same principle of directed growth by a motor protein connected to a growing microtubule plus end could be used to align microtubules in many circumstances (Mattie, 2010).
Similar to mammalian APCs, Drosophila Apc contains several potential glycosylation sites and phosphorylation sites and lacks a signal sequence or transmembrane domain. The deduced Apc protein has 27% identity and 46% similarity over all to human APC. Furthermore, there is striking homology in domains previously identified in m-APC: like m-APCs, the amino-terminal third of Apc contains seven armadillo repeats with approximately 60% identity to those observed in the human protein. The repeats lie outside the region recognized by the human antibody used to screen the expression library; their presence provides further support that the gene described here is an APC homolog. In addition, Apc has at least 6 of the 10 beta-catenin binding sites previously identified in human APC, with ~50% identity at the amino acid level. Apc contains both types of beta-catenin binding sites observed in the human protein, one 15-amino acid repeat with the PXXYS motif thought to bind beta-catenin, and 5 of the 20-amino acid repeats with the PXXFS motif thought to provide beta-catenin down-regulating activity. Such repeats are not found in other beta-catenin binding proteins, such as E-cadherin or alpha-catenin, and are thought to be hallmarks of m-APCs. There are two additional regions in Apc that contain a PXXXS motif. One of these regions shows 40-50% identity to various 20-amino acid repeats, and the other region shows 40-50% identity to various beta-catenin binding sites and 20-amino acid repeats in the human protein, though these repeats do not align with any particular mammalian repeat. One of the regions with the PXXFS motif in Apc could be a 15-amino acid repeat, based on its alignment to the second 15-amino acid repeat of human APC. Because their exact function is not known, one of the PXXFS motifs and two PXXXS motif sites could raise the number of beta-catenin binding sites (15-amino acid repeat) from one to three, or the number of 20-amino acid repeats from five to seven. A potential GSK (Shaggy/zeste white 3) phosphorylation site has been observed in five out of seven 20-amino acid repeats. These results also suggest that overall homology between Apc and m-APCs could be underestimated due to lack of alignment in some conserved domains.
The carboxy terminus of Drosophila Apc and human APC do not show significant homology -- 21% amino acid identity in the carboxyl-terminal 72 amino acids containing the Discs large (Dlg) binding site. At carboxyl-terminal end, Apc lacks the S/T(X)V motif, a sequence required for the binding to the Discs large homology repeat domain. However, the carboxy terminal 270 amino acids of Apc show a charged basic character as observed in m-APCs between amino acids 2200 and 2400. This basic region has been implicated in the binding of m-APCs to microtubules. The basic domain of Apc has weak homology to the microtubule binding domain of microtubule-associated protein 4. This finding suggests that the carboxy terminus of Drosophila Apc may function like that of human APC in binding to microtubules. In addition to the domains mentioned above, a well conserved region of amino acids not previously identified as a separate entity is located slightly amino terminal to the armadillo repeats between amino acids 308 and 417. This conserved domain has 50% amino acid identity between Drosophila Apc and m-APCs, indicating an interesting region yet to be studied functionally. A very limited homology of Apc to m-APCs was observed in the oligomerization domain, as well as the Discs large binding site. This finding does not exclude the possibility that Apc may dimerize or interact using a different site. It remains possible that alternative splicing might produce as-yet-undetected Apc variants with high homology to m-APCs in this region. Alternative splicing forms of the cDNA in 5' end of human APC have been reported. Altogether, this similarity of domain structure of Drosophila Apc to m-APCs, including armadillo repeats, beta-catenin binding sites, and the basic domain suggests that Apc may have significant functional similarity to m-APCs (Hayashi, 1997).
date revised: 15 July 98
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