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. 2019 Aug 2;39(8):BSR20180604.
doi: 10.1042/BSR20180604. Print 2019 Aug 30.

Armc8 is an evolutionarily conserved armadillo protein involved in cell-cell adhesion complexes through multiple molecular interactions

Ismail Sahin Gul  1   2 , Paco Hulpiau  1   2   3 , Ellen Sanders  1   2 , Frans van Roy  1   2 , Jolanda van Hengel  4
Affiliations

Armc8 is an evolutionarily conserved armadillo protein involved in cell-cell adhesion complexes through multiple molecular interactions

Ismail Sahin Gul et al. Biosci Rep. .

Abstract

Armadillo-repeat-containing protein 8 (Armc8) belongs to the family of armadillo-repeat containing proteins, which have been found to be involved in diverse cellular functions including cell-cell contacts and intracellular signaling. By comparative analyses of armadillo repeat protein structures and genomes from various premetazoan and metazoan species, we identified orthologs of human Armc8 and analyzed in detail the evolutionary relationship of Armc8 genes and their encoded proteins. Armc8 is a highly ancestral armadillo protein although not present in yeast. Consequently, Armc8 is not the human ortholog of yeast Gid5/Vid28.Further, we performed a candidate approach to characterize new protein interactors of Armc8. Interactions between Armc8 and specific δ-catenins (plakophilins-1, -2, -3 and p0071) were observed by the yeast two-hybrid approach and confirmed by co-immunoprecipitation and co-localization. We also showed that Armc8 interacts specifically with αE-catenin but neither with αN-catenin nor with αT-catenin. Degradation of αE-catenin has been reported to be important in cancer and to be regulated by Armc8. A similar process may occur with respect to plakophilins in desmosomes. Deregulation of desmosomal proteins has been considered to contribute to tumorigenesis.

Keywords: Armadillo protein; Armc8; desmosome; molecular evolution.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1
Figure 1. Yeast two-hybrid (Y2H) assay to study the possible interactions between human Armc8α and members of the α- and δ-catenin protein families
(A) Schematic representation of the Armc8 protein constructs used. Armadillo repeats are represented by red cylinders. (B–D) The Matchmaker Gold yeast strain Y2H Gold was co-transformed with different expression plasmids. All transformations shown were successful since transformed yeasts grew on SD-LT (L = leucine; T = tryptophan) selective medium (left panels, -LT). Data are each time shown as three representative yeast colonies. The Y2H Gold strain allows reliable detection of one-to-one protein interactions as the rescued GAL4 expression activates the selection and reporter genes ADE2, HIS3 and MEL1 (α-galactosidase). Positive interactions yield blue colonies on SD–LTHA (H = histidine; A = adenine) containing X-α-Galactosidase (right panels, -LTHA + XαGal). All results were reproduced in at least three biological replicates. For all bait and prey vectors used, a test for auto-activation was negative. (B) Testing the interaction between full-length human Armc8α or a C-terminal Armc8α fragment against the three α-catenin paralogs identified only αE-catenin as interaction partner of Armc8α (C-term). (C) Testing the interaction between the two Armc8α constructs against plakophilins showed that the Armc8α (C-term) could interact with all plakophilins. (D) Testing the interaction between the two Armc8α constructs against the CTNND core proteins identified p0071 but not the other armadillo proteins as interaction partner of Armc8α (C-term).
Figure 2
Figure 2. Phylogenetic analysis of identified Armc8 orthologs and of armadillo catenins
Protein sequences of human Armc8α and its orthologs in metazoan and non-metazoan species (see Table 1) were aligned mutually and with human, California sea hare (Aplysia californica) and placozoan (Trichoplax adhaerens) armadillo catenin proteins. The phylogenetic analysis was performed with the neighbor-joining method and bootstrap values were provided for each branch. The phylogenetic tree was visualized using Dendroscope [40].
Figure 3
Figure 3. Synteny of ARMC8 and flanking genes in chordates
Comparison of the syntenic blocks shared between human chromosome 3q22.3 and corresponding chromosomal regions in four other vertebrates (Mus musculus, Gallus gallus, Xenopus tropicalis and Danio rerio) and the chordate Branchiostoma floridae. In each genomic region analyzed, ARMC8 is represented by a blue arrow.
Figure 4
Figure 4. Alignment and helical wheel representation of the α-catenin binding regions in human β-catenin and Armc8α
(A) Alignment of human β-catenin region AA 121–165 and human Armc8α region AA 296–339 shows the sequence similarity of both α-catenin binding regions. (B) Left, characterized binding helices in human β-catenin. The yellow ellipse highlights the evolutionarily conserved surface that binds αE-catenin [25]. Right, predicted α-catenin binding region in human Armc8α; the yellow ellipse highlights the evolutionarily conserved putative surface binding αE-catenin. Similar AA in the predicted α-catenin binding regions are indicated with red stars. Detailed results of the prediction to support this are included as Supplementary Figure S2.
Figure 5
Figure 5. Y2H assay to narrow down the interactions between Armc8, Pkp3 and p0071 proteins
(A) Schematic structures of Armc8α, Armc8β, Pkp3, p0071 and their truncated forms used in this experiment. Armadillo repeats are represented by red cylinders. (B) Testing the interaction between Armc8α and Armc8β constructs against the N- and C-terminally truncated p0071 proteins characterized the tail region of p0071 as binding region to both Armc8α (C-term) and Armc8β. (C) Testing the interaction between Armc8α and Armc8β constructs against the full-length Pkp3 and a truncated derivative suggests that the N-terminal domain of Pkp3 is required for the interaction with a region shared by Armc8α and Armc8β.
Figure 6
Figure 6. Association of endogenous Armc8α and Pkp3 in SKCO-15 cells
(A) Endogenous Pkp3 was co-immunoprecipitated with Armc8 from a SKCO-15 cell lysate. The specificity of the antibodies used is as indicated. Initial immunoprecipation (IP) was followed by detection of Pkp3 in a Western blot (WB) of the immunoprecipitates. No interaction was observed in the control immunoprecipitation with an anti-Flag antibody. (B) Orthographic and planar projections were obtained by confocal microscopy. Images of SKCO-15 cells showing co-localiation of endogenous Armc8 (red) and Pkp3 (green) at cell–cell contacts. In the lower panel, regions of co-localization appear as yellow. Specific monoclonal antibodies were used; scale bars = 10 µm.
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References

    1. Tewari R., Bailes E., Bunting K.A. and Coates J.C. (2010) Armadillo-repeat protein functions: questions for little creatures. Trends Cell Biol. 20, 470–48110.1016/j.tcb.2010.05.003 - DOI - PubMed
    1. Gul I.S., Hulpiau P., Saeys Y. and van Roy F. (2017) Metazoan evolution of the armadillo repeat superfamily. Cell. Mol. Life Sci. 74, 525–54110.1007/s00018-016-2319-6 - DOI - PMC - PubMed
    1. Kobayashi N., Yang J., Ueda A., Suzuki T., Tomaru K., Takeno M.. et al. (2007) RanBPM, Muskelin, p48EMLP, p44CTLH, and the armadillo-repeat proteins ARMC8alpha and ARMC8beta are components of the CTLH complex. Gene 396, 236–24710.1016/j.gene.2007.02.032 - DOI - PubMed
    1. Santt O., Pfirrmann T., Braun B., Juretschke J., Kimmig P., Scheel H.. et al. (2008) The yeast GID complex, a novel ubiquitin ligase (E3) involved in the regulation of carbohydrate metabolism. Mol. Biol. Cell 19, 3323–333310.1091/mbc.e08-03-0328 - DOI - PMC - PubMed
    1. Giardina B.J., Dunton D. and Chiang H.L. (2013) Vid28 protein is required for the association of vacuole import and degradation (Vid) vesicles with actin patches and the retention of Vid vesicle proteins in the intracellular fraction. J. Biol. Chem. 288, 11636–1164810.1074/jbc.M112.419895 - DOI - PMC - PubMed

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