Drosophila Naked cuticle (Nkd) engages the nuclear import adaptor Importin-α3 to antagonize Wnt/β-catenin signaling

Laboratory of Molecular Pathology, Department of Pathology, UT Southwestern Medical Center, Dallas, TX 75390-9072, USA.
Developmental Biology (Impact Factor: 3.55). 07/2008; 318(1):17-28. DOI: 10.1016/j.ydbio.2008.02.050
Source: PubMed


Precise control of Wnt/beta-catenin signaling is critical for animal development, stem cell renewal, and prevention of disease. In the fruit fly Drosophila melanogaster, the naked cuticle (nkd) gene limits signaling by the Wnt ligand Wingless (Wg) during embryo segmentation. Nkd is an intracellular protein that is composed of separable membrane- and nuclear-localization sequences (NLS) as well as a conserved EF-hand motif that binds the Wnt receptor-associated scaffold protein Dishevelled (Dsh), but the mechanism by which Nkd inhibits Wnt signaling remains a mystery. Here we identify a second NLS in Nkd that is required for full activity and that binds to the canonical nuclear import adaptor Importin-alpha3. The Nkd NLS is similar to the Importin-alpha3-binding NLS in the Drosophila heat-shock transcription factor (dHSF), and each Importin-alpha3-binding NLS required intact basic residues in similar positions for nuclear import and protein function. Our results provide further support for the hypothesis that Nkd inhibits nuclear step(s) in Wnt/beta-catenin signaling and broaden our understanding of signaling pathways that engage the nuclear import machinery.

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    • "In an effort to identify novel components involved in Notch signaling and its regulation, a yeast two-hybrid screen was carried out using the portion of intracellular domain of Notch receptor as bait and we identified Drosophila Importin-α3 as binding partner of Notch. Drosophila Importin-α3 protein is known to play major role in nuclear trafficking of different Nuclear Localization Signal (NLS) containing proteins such as Germ Cell-less [18], the large subunit of DNA polymerase α [19], heat shock transcription factor (dHSF) [20], Daxx [21], Naked cuticle (Nkd) [22] etc. Since nuclear transport protein Importin-α3 directly binds to portion of Notch intracellular domain which contains NLS [7], we were prompted to examine if Notch intracellular domain translocates to nucleus using the canonical nuclear transport machinery. "
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    ABSTRACT: The Notch signaling pathway controls diverse cell-fate specification events throughout development. The versatility of this pathway to influence different aspects of development comes from its multiple levels of regulation. Upon ligand-induced Notch activation, the Notch intracellular domain (Notch-ICD) is released from the membrane and translocates to the nucleus, where it transduces Notch signals by regulating the transcription of downstream target genes. But the exact mechanism of translocation of Notch-ICD into the nucleus is not clear. Here, we implicate Importin-α3 (also known as karyopherin-α3) in the nuclear translocation of Notch-ICD in Drosophila. Our present analyses reveal that Importin-α3 can directly bind to Notch-ICD and loss of Importin-α3 function results in cytoplasmic accumulation of the Notch receptor. Using MARCM (Mosaic Analysis with a Repressible Cell Marker) technique, we demonstrate that Importin-α3 is required for nuclear localization of Notch-ICD. These results reveal that the nuclear transport of Notch-ICD is mediated by the canonical Importin-α3/Importin-β transport pathway. In addition, co-expression of both Notch-ICD and Importin-α3 displays synergistic effects on cell proliferation. Taken together, our results suggest that Importin-α3 mediated nuclear import of Notch-ICD may play important role in regulation of Notch signaling.
    PLoS ONE 07/2013; 8(7):e68247. DOI:10.1371/journal.pone.0068247 · 3.23 Impact Factor
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    • "Immunostaining of salivary glands from 71B>impα1RNAi, 71B>impα2RNAi and 71B>impα3RNAi larvae revealed no changes in Cbt nuclear localization (Figure S3A–D). However, only the impα3RNAi line has been shown to reduce Impα3 immunoreactivity [77]. "
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    ABSTRACT: Cabut (Cbt) is a C(2)H(2)-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. To determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using β-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2. Our results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins.
    PLoS ONE 02/2012; 7(2):e32004. DOI:10.1371/journal.pone.0032004 · 3.23 Impact Factor
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    • "In abundance of other Dvl PDZ binding partners, the NES motif would be released making the protein more susceptible to nuclear export and resulting in an increased induction of PCP and/or calcium signaling cascade. So far, our understanding of the Wnt signaling network is very limited, and we are still discovering new possible regulatory mechanisms (Yokoyama et al., 2007; Chan et al., 2008; Wu et al., 2008) and links to other cellular processes and pathways (Luyten et al., 2008) to broaden our comprehension of the molecular basis of development. "
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    ABSTRACT: Intracellular signaling cascades induced by Wnt proteins play a key role in developmental processes and are implicated in cancerogenesis. It is still unclear how the cell determines which of the three possible Wnt response mechanisms should be activated, but the decision process is most likely dependent on Dishevelled proteins. Dishevelled family members interact with many diverse targets, however, molecular mechanisms underlying these binding events have not been comprehensively described so far. Here, we investigated the specificity of the PDZ domain from human Dishevelled-2 using C-terminal phage display, which led us to identification of a leucine-rich binding motif strongly resembling the consensus sequence of a nuclear export signal. PDZ interactions with several peptide and protein motifs (including the nuclear export signal sequence from Dishevelled-2 protein) were investigated in detail using fluorescence spectroscopy, mutational analysis and immunoenzymatic assays. The experiments showed that the PDZ domain can bind the nuclear export signal sequence of the Dishevelled-2 protein. Since the intracellular localization of Dishevelled is governed by nuclear localization and nuclear export signal sequences, it is possible that the intramolecular interaction between PDZ domain and the export signal could modulate the balance between nuclear and cytoplasmic pool of the Dishevelled protein. Such a regulatory mechanism would be of utmost importance for the differential activation of Wnt signaling cascades, leading to selective promotion of the nucleus-dependent Wnt β-catenin pathway at the expense of non-canonical Wnt signaling.
    Acta biochimica Polonica 01/2011; 58(2):243-9. · 1.15 Impact Factor
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