Drosophila Apc2 Is a Cytoskeletally-Associated Protein That Regulates Wingless Signaling in the Embryonic Epidermis

Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3280, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 10/1999; 146(6):1303-18. DOI: 10.1083/jcb.146.6.1303
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The tumor suppressor adenomatous polyposis coli (APC) negatively regulates Wingless (Wg)/Wnt signal transduction by helping target the Wnt effector beta-catenin or its Drosophila homologue Armadillo (Arm) for destruction. In cultured mammalian cells, APC localizes to the cell cortex near the ends of microtubules. Drosophila APC (dAPC) negatively regulates Arm signaling, but only in a limited set of tissues. We describe a second fly APC, dAPC2, which binds Arm and is expressed in a broad spectrum of tissues. dAPC2's subcellular localization revealed colocalization with actin in many but not all cellular contexts, and also suggested a possible interaction with astral microtubules. For example, dAPC2 has a striking asymmetric distribution in neuroblasts, and dAPC2 colocalizes with assembling actin filaments at the base of developing larval denticles. We identified a dAPC2 mutation, revealing that dAPC2 is a negative regulator of Wg signaling in the embryonic epidermis. This allele acts genetically downstream of wg, and upstream of arm, dTCF, and, surprisingly, dishevelled. We discuss the implications of our results for Wg signaling, and suggest a role for dAPC2 as a mediator of Wg effects on the cytoskeleton. We also speculate on more general roles that APCs may play in cytoskeletal dynamics.

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Available from: Catherine A Kirkpatrick, May 31, 2014
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    • "Among these, patterning of the embryonic epidermis and the wing imaginal disc has been extensively studied to understand the mechanism of Wg signaling. In embryogenesis, Wg is expressed in a strip of cells within each segmental unit and is required for anterior-posterior patterning of the epidermis (van den Heuvel et al., 1993; Siegfried et al., 1994; McCartney et al., 1999). "
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    ABSTRACT: Wingless (Wg)/Wnt signaling is fundamental in metazoan development. Armadillo (Arm)/β-catenin and Dishevelled (Dsh) are key components of Wnt signal transduction. Recent studies suggest that intracellular trafficking of Wnt signaling components is important, but underlying mechanisms are not well known. Here, we show that Klp64D, the Drosophila homolog of Kif3A kinesin II subunit, is required for Wg signaling by regulating Arm during wing development. Mutations in klp64D or RNAi cause wing notching and loss of Wg target gene expression. The wing notching phenotype by Klp64D knockdown is suppressed by activated Arm but not by Dsh, suggesting that Klp64D is required for Arm function. Furthermore, klp64D and arm mutants show synergistic genetic interaction. Consistent with this genetic interaction, Klp64D directly binds to the Arm repeat domain of Arm and can recruit Dsh in the presence of Arm. Overexpression of Klp64D mutated in the motor domain causes dominant wing notching, indicating the importance of the motor activity. Klp64D shows subcellular localization to intracellular vesicles overlapping with Arm and Dsh. In klp64D mutants, Arm is abnormally accumulated in vesicular structures including Golgi, suggesting that intracellular trafficking of Arm is affected. Human KIF3A can also bind β-catenin and rescue klp64D RNAi phenotypes. Taken together, we propose that Klp64D is essential for Wg signaling by trafficking of Arm via the formation of a conserved complex with Arm.
    Journal of Cell Science 07/2014; 141(16). DOI:10.1242/dev.106229 · 5.43 Impact Factor
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    • "Despite its essential roles in regulating stem cell self-renewal and, ultimately, the generation of differentiated cells, little is known about the role of Wnt signaling in the specification of progenitor cell identity. Previous studies revealed that Drosophila Apc2 is cortically enriched asymmetrically in larval brain neuroblasts, such that a portion of the Apc2 is partitioned into the progenitor cell (McCartney et al., 1999). Furthermore, Apc2 becomes enriched in the cortex of the progenitor cell following neuroblast asymmetric division, suggesting that Wnt signaling activity might be negatively regulated in the progenitor cell (Akong et al., 2002). "
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    ABSTRACT: During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. Here, we report that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/Armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the self-renewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification.
    Development 11/2013; 141(1). DOI:10.1242/dev.099382 · 6.46 Impact Factor
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    • "The middle third of APC carries a series of short binding sites for proteins involved in Wnt regulation, including 15- and 20-amino acid repeats (15R and 20R), which bind ß-catenin and SAMP repeats, which bind Axin. It also contains the short conserved sequence B [31], also known as the Catenin inhibitory domain (CID) [32]. Proposed models also consider the fact that both APC and Axin have ß-catenin binding sites, but unlike APC, Axin has a single ß-catenin binding site. "
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    ABSTRACT: The tumor suppressor Adenomatous Polyposis coli (APC) gene is mutated or lost in most colon cancers. Alterations in Protein kinase C (PKC) isozyme expression and aberrant regulation also comprise early events in intestinal carcinomas. Here we show that PKCδ expression levels are decreased in colon tumor cell lines with respect to non-malignant cells. Reciprocal co-immunoprecipitation and immunofluorescence studies revealed that PKCδ interacts specifically with both full-length (from non-malignant cells) and truncated APC protein (from cancerous cells) at the cytoplasm and at the cell nucleus. Selective inhibition of PKCδ in cancer SW480 cells, which do not possess a functional β-catenin destruction complex, did not affect β-catenin-mediated transcriptional activity. However, in human colon carcinoma RKO cells, which have a normal β-catenin destruction complex, negatively affected β-catenin-mediated transcriptional activity, cell proliferation, and the expression of Wnt target genes C-MYC and CYCLIN D1. These negative effects were confirmed by siRNA-mediated knockdown of PKCδ and by the expression of a dominant negative form of PKCδ in RKO cells. Remarkably, the PKCδ stably depleted cells exhibited augmented tumorigenic activity in grafted mice. We show that PKCδ functions in a mechanism that involves regulation of β-catenin degradation, because PKCδ inhibition induces β-catenin stabilization at the cytoplasm and its nuclear presence at the C-MYC enhancer even without Wnt3a stimulation. In addition, expression of a dominant form of PKCδ diminished APC phosphorylation in intact cells, suggesting that PKCδ may modulate canonical Wnt activation negatively through APC phosphorylation.
    PLoS ONE 03/2013; 8(3):e58540. DOI:10.1371/journal.pone.0058540 · 3.23 Impact Factor
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