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Defining desmosomal plakophilin-3 interactions.

Bonné S, Gilbert B, Hatzfeld M, Chen X, Green KJ, van Roy F - J. Cell Biol. (2003)

Bottom Line: We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a.Evidence was found for the presence of at least two DP-PKP3 interaction sites.Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology Unit, Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology (VIB)-Ghent University, B-9000 Ghent, Belgium.

ABSTRACT
Plakophilin 3 (PKP3) is a recently described armadillo protein of the desmosomal plaque, which is synthesized in simple and stratified epithelia. We investigated the localization pattern of endogenous and exogenous PKP3 and fragments thereof. The desmosomal binding properties of PKP3 were determined using yeast two-hybrid, coimmunoprecipitation and colocalization experiments. To this end, novel mouse anti-PKP3 mAbs were generated. We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a. As such, this is the first protein interaction ever observed with a Dsc-b isoform. Moreover, we determined that PKP3 interacts with plakoglobin, desmoplakin (DP) and the epithelial keratin 18. Evidence was found for the presence of at least two DP-PKP3 interaction sites. This finding might explain how lateral DP-PKP interactions are established in the upper layers of stratified epithelia, increasing the size of the desmosome and the number of anchoring points available for keratins. Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.

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Localization of desmosome-related proteins in various transfected cells. GFP- and FLAG-tagged proteins wre expressed in MCF7/AZ (a–f), HCT8/E8 (g–i), COS1 (j–l), and COS7 (m–o) cells. PKP3head (a and j) and PKP3headΔHR2 (d and g) easily accumulate as discrete cytoplasmic aggregates (arrows in a and g). Endogenous DP (b) localizes to cell–cell contacts (arrowheads) and aggregates (arrows). There is clear colocalization with GFP-tagged PKP3head in these aggregates (c). Insets represent larger magnifications of the areas indicated with arrows. In contrast, GFP-tagged PKP3headΔHR2 (d) and DP (e) do not colocalize (f), whereas GFP-tagged PKP3ΔHR2 (g) and CK18 (h) still localize together in aggregates (i), as indicated by arrows (also see insets, representing larger magnifications). The same observation is made for PKP3head in COS1 cells (j–l). Oversynthesis of PKP3headΔHR2 or PKP3head results in disturbance of the keratin intermediate filament network as revealed by the anti-CK18 antibody (h and k). Single transfection of a DP.FLAG construct in COS7 results in decoration of the keratin network and a punctate localization along cell–cell contacts of the exogenous DP.FLAG protein (m). In DP.FLAG and PKP3GFP cotransfected cells, the DP.FLAG localization along cell contacts is more continuous (n), and DP.FLAG and PKP3GFP colocalize in these structures (o). Bars: 20 μm (a–c, m) and 10 μm (d–l, n–o).
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fig6: Localization of desmosome-related proteins in various transfected cells. GFP- and FLAG-tagged proteins wre expressed in MCF7/AZ (a–f), HCT8/E8 (g–i), COS1 (j–l), and COS7 (m–o) cells. PKP3head (a and j) and PKP3headΔHR2 (d and g) easily accumulate as discrete cytoplasmic aggregates (arrows in a and g). Endogenous DP (b) localizes to cell–cell contacts (arrowheads) and aggregates (arrows). There is clear colocalization with GFP-tagged PKP3head in these aggregates (c). Insets represent larger magnifications of the areas indicated with arrows. In contrast, GFP-tagged PKP3headΔHR2 (d) and DP (e) do not colocalize (f), whereas GFP-tagged PKP3ΔHR2 (g) and CK18 (h) still localize together in aggregates (i), as indicated by arrows (also see insets, representing larger magnifications). The same observation is made for PKP3head in COS1 cells (j–l). Oversynthesis of PKP3headΔHR2 or PKP3head results in disturbance of the keratin intermediate filament network as revealed by the anti-CK18 antibody (h and k). Single transfection of a DP.FLAG construct in COS7 results in decoration of the keratin network and a punctate localization along cell–cell contacts of the exogenous DP.FLAG protein (m). In DP.FLAG and PKP3GFP cotransfected cells, the DP.FLAG localization along cell contacts is more continuous (n), and DP.FLAG and PKP3GFP colocalize in these structures (o). Bars: 20 μm (a–c, m) and 10 μm (d–l, n–o).

Mentions: MCF7/AZ, HCT8/E8, and COS cells were transfected with constructs encoding GFP-tagged PKP3 or fragments thereof and then subjected to immunofluorescence detection of desmosome-related proteins. PKP3head localized in MCF7/AZ cells as described in the previous paragraph (illustrated in Fig. 6 a). Immunodetection revealed DP not only to be present at sites of cell–cell contacts in MCF7/AZ cells, but also in cytoplasmic aggregates (Fig. 6 b), where it colocalizes with PKP3head (Fig. 6 c). This observation was more clear in MCF7/AZ cells than in HCT8/E8 cells (compare with Fig. 4, i and j). DP did not colocalize with GFP-tagged PKP3headΔHR2 in aggregates, as it was only detected in desmosomal cell–cell contacts (Fig. 6, d–f). This observation suggests that the HR2 domain is involved in PKP3 binding to DP. In contrast, CK18 is observed in aggregates containing either GFP-tagged PKP3head (unpublished data) or GFP-tagged PKP3headΔHR2 protein (Fig. 6, g–i), suggesting that the head domain of PKP3 (but not the HR2 domain) is involved in CK18 interaction. Apparently, oversynthesis of these head fragments results in disturbance of the keratin filament network. Both DP and CK18 bind the PKP3head domain in yeast two-hybrid experiments (see following paragraph). Other desmosomal proteins, like Dsg2 and PKP2, were not found to colocalize in PKP3head aggregates (unpublished data). Similar results were obtained using COS1 cells in transfection experiments, as exemplified by the association of CK18 with PKP3headGFP in COS1 cells (Fig. 6, j–l).


Defining desmosomal plakophilin-3 interactions.

Bonné S, Gilbert B, Hatzfeld M, Chen X, Green KJ, van Roy F - J. Cell Biol. (2003)

Localization of desmosome-related proteins in various transfected cells. GFP- and FLAG-tagged proteins wre expressed in MCF7/AZ (a–f), HCT8/E8 (g–i), COS1 (j–l), and COS7 (m–o) cells. PKP3head (a and j) and PKP3headΔHR2 (d and g) easily accumulate as discrete cytoplasmic aggregates (arrows in a and g). Endogenous DP (b) localizes to cell–cell contacts (arrowheads) and aggregates (arrows). There is clear colocalization with GFP-tagged PKP3head in these aggregates (c). Insets represent larger magnifications of the areas indicated with arrows. In contrast, GFP-tagged PKP3headΔHR2 (d) and DP (e) do not colocalize (f), whereas GFP-tagged PKP3ΔHR2 (g) and CK18 (h) still localize together in aggregates (i), as indicated by arrows (also see insets, representing larger magnifications). The same observation is made for PKP3head in COS1 cells (j–l). Oversynthesis of PKP3headΔHR2 or PKP3head results in disturbance of the keratin intermediate filament network as revealed by the anti-CK18 antibody (h and k). Single transfection of a DP.FLAG construct in COS7 results in decoration of the keratin network and a punctate localization along cell–cell contacts of the exogenous DP.FLAG protein (m). In DP.FLAG and PKP3GFP cotransfected cells, the DP.FLAG localization along cell contacts is more continuous (n), and DP.FLAG and PKP3GFP colocalize in these structures (o). Bars: 20 μm (a–c, m) and 10 μm (d–l, n–o).
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fig6: Localization of desmosome-related proteins in various transfected cells. GFP- and FLAG-tagged proteins wre expressed in MCF7/AZ (a–f), HCT8/E8 (g–i), COS1 (j–l), and COS7 (m–o) cells. PKP3head (a and j) and PKP3headΔHR2 (d and g) easily accumulate as discrete cytoplasmic aggregates (arrows in a and g). Endogenous DP (b) localizes to cell–cell contacts (arrowheads) and aggregates (arrows). There is clear colocalization with GFP-tagged PKP3head in these aggregates (c). Insets represent larger magnifications of the areas indicated with arrows. In contrast, GFP-tagged PKP3headΔHR2 (d) and DP (e) do not colocalize (f), whereas GFP-tagged PKP3ΔHR2 (g) and CK18 (h) still localize together in aggregates (i), as indicated by arrows (also see insets, representing larger magnifications). The same observation is made for PKP3head in COS1 cells (j–l). Oversynthesis of PKP3headΔHR2 or PKP3head results in disturbance of the keratin intermediate filament network as revealed by the anti-CK18 antibody (h and k). Single transfection of a DP.FLAG construct in COS7 results in decoration of the keratin network and a punctate localization along cell–cell contacts of the exogenous DP.FLAG protein (m). In DP.FLAG and PKP3GFP cotransfected cells, the DP.FLAG localization along cell contacts is more continuous (n), and DP.FLAG and PKP3GFP colocalize in these structures (o). Bars: 20 μm (a–c, m) and 10 μm (d–l, n–o).
Mentions: MCF7/AZ, HCT8/E8, and COS cells were transfected with constructs encoding GFP-tagged PKP3 or fragments thereof and then subjected to immunofluorescence detection of desmosome-related proteins. PKP3head localized in MCF7/AZ cells as described in the previous paragraph (illustrated in Fig. 6 a). Immunodetection revealed DP not only to be present at sites of cell–cell contacts in MCF7/AZ cells, but also in cytoplasmic aggregates (Fig. 6 b), where it colocalizes with PKP3head (Fig. 6 c). This observation was more clear in MCF7/AZ cells than in HCT8/E8 cells (compare with Fig. 4, i and j). DP did not colocalize with GFP-tagged PKP3headΔHR2 in aggregates, as it was only detected in desmosomal cell–cell contacts (Fig. 6, d–f). This observation suggests that the HR2 domain is involved in PKP3 binding to DP. In contrast, CK18 is observed in aggregates containing either GFP-tagged PKP3head (unpublished data) or GFP-tagged PKP3headΔHR2 protein (Fig. 6, g–i), suggesting that the head domain of PKP3 (but not the HR2 domain) is involved in CK18 interaction. Apparently, oversynthesis of these head fragments results in disturbance of the keratin filament network. Both DP and CK18 bind the PKP3head domain in yeast two-hybrid experiments (see following paragraph). Other desmosomal proteins, like Dsg2 and PKP2, were not found to colocalize in PKP3head aggregates (unpublished data). Similar results were obtained using COS1 cells in transfection experiments, as exemplified by the association of CK18 with PKP3headGFP in COS1 cells (Fig. 6, j–l).

Bottom Line: We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a.Evidence was found for the presence of at least two DP-PKP3 interaction sites.Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cell Biology Unit, Department for Molecular Biomedical Research, Flanders Interuniversity Institute for Biotechnology (VIB)-Ghent University, B-9000 Ghent, Belgium.

ABSTRACT
Plakophilin 3 (PKP3) is a recently described armadillo protein of the desmosomal plaque, which is synthesized in simple and stratified epithelia. We investigated the localization pattern of endogenous and exogenous PKP3 and fragments thereof. The desmosomal binding properties of PKP3 were determined using yeast two-hybrid, coimmunoprecipitation and colocalization experiments. To this end, novel mouse anti-PKP3 mAbs were generated. We found that PKP3 binds all three desmogleins, desmocollin (Dsc) 3a and -3b, and possibly also Dsc1a and -2a. As such, this is the first protein interaction ever observed with a Dsc-b isoform. Moreover, we determined that PKP3 interacts with plakoglobin, desmoplakin (DP) and the epithelial keratin 18. Evidence was found for the presence of at least two DP-PKP3 interaction sites. This finding might explain how lateral DP-PKP interactions are established in the upper layers of stratified epithelia, increasing the size of the desmosome and the number of anchoring points available for keratins. Together, these results show that PKP3, whose epithelial and epidermal desmosomal expression pattern and protein interaction repertoire are broader than those of PKP1 and -2, is a unique multiprotein binding element in the basic architecture of a vast majority of epithelial desmosomes.

Show MeSH
Related in: MedlinePlus