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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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Intracellular localization of exogenous GFP-tagged PKP3 and fragments thereof in HCT8/E8 and MCF7/AZ cells. In HCT8/E8 cells, full-length GFP-tagged PKP3 localizes predominantly in desmosomes and to a lesser extent in the cytoplasm (a). PKP3GFP and DP (b) overlap in desmosomes of transfected cells (c). The punctate localization of these proteins along cell–cell contacts is clear from the larger magnifications (a′–c′) of the fields indicated by arrowheads in pictures a–c. GFP-tagged PKP3ΔHR2 displays a similar intracellular localization as the full-length PKP3 (d), and colocalizes with DP (e) in desmosomes of transfected cells (f). The punctate localization of these proteins along cell–cell contacts is obvious from the larger magnifications (d′–f′) of the fields indicated by arrowheads in pictures d–f. As is clear from images d′′–f′′ (fields indicated by arrows in pictures d–f), GFP-tagged PKP3 overlaps only half of the DP signal if transfected cells contact untransfected cells. PKP3arm GFP accumulates to high levels in the cell nucleus, but almost not at all at cell–cell contacts (g); control immunodetection of DP in the same cell field (h). GFP-tagged PKP3head protein accumulates as discrete cytoplasmic aggregates, in addition to a more diffuse cytoplasmic localization and less intense accumulation at cell–cell contacts (i); control detection of DP in the same field (j). GFP-tagged PKP3head is observed in a similar pattern in MCF7/AZ cells (k); endogenous PKP3 is detected using antibody 23E3/4 (l). Arrowheads, occasional endogenous PKP3 localization in PKP3headGFP aggregates; arrows, colocalization of exogenous fusion protein and endogenous PKP3 at sites of cell–cell contact. The marked regions are enlarged in the insets. Bars: 10 μm (a–j) and 20 μm (k and l).
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fig4: Intracellular localization of exogenous GFP-tagged PKP3 and fragments thereof in HCT8/E8 and MCF7/AZ cells. In HCT8/E8 cells, full-length GFP-tagged PKP3 localizes predominantly in desmosomes and to a lesser extent in the cytoplasm (a). PKP3GFP and DP (b) overlap in desmosomes of transfected cells (c). The punctate localization of these proteins along cell–cell contacts is clear from the larger magnifications (a′–c′) of the fields indicated by arrowheads in pictures a–c. GFP-tagged PKP3ΔHR2 displays a similar intracellular localization as the full-length PKP3 (d), and colocalizes with DP (e) in desmosomes of transfected cells (f). The punctate localization of these proteins along cell–cell contacts is obvious from the larger magnifications (d′–f′) of the fields indicated by arrowheads in pictures d–f. As is clear from images d′′–f′′ (fields indicated by arrows in pictures d–f), GFP-tagged PKP3 overlaps only half of the DP signal if transfected cells contact untransfected cells. PKP3arm GFP accumulates to high levels in the cell nucleus, but almost not at all at cell–cell contacts (g); control immunodetection of DP in the same cell field (h). GFP-tagged PKP3head protein accumulates as discrete cytoplasmic aggregates, in addition to a more diffuse cytoplasmic localization and less intense accumulation at cell–cell contacts (i); control detection of DP in the same field (j). GFP-tagged PKP3head is observed in a similar pattern in MCF7/AZ cells (k); endogenous PKP3 is detected using antibody 23E3/4 (l). Arrowheads, occasional endogenous PKP3 localization in PKP3headGFP aggregates; arrows, colocalization of exogenous fusion protein and endogenous PKP3 at sites of cell–cell contact. The marked regions are enlarged in the insets. Bars: 10 μm (a–j) and 20 μm (k and l).

Mentions: To study the intracellular distribution of full-length PKP3 and fragments thereof, transfection of PKP3 expression constructs was performed in HCT8/E8, MCF7/AZ, COS1, and PtK2 cells, yielding consistent data. A schematic overview of the protein fragments encoded by the different constructs is shown in Fig. 3 . In HCT8/E8 cells, full-length human PKP3 tagged with GFP displays an intracellular distribution similar to endogenous PKP3 detected by mAbs, i.e., a clear-cut localization in desmosomes, weaker in the cytoplasm, and absent from the cell nucleus (Fig. 4 a). Exogenous PKP3 colocalizes with endogenous DP in desmosomes (Fig. 4, b and c). Larger magnifications emphasize the punctate (co)localization of these proteins along cell–cell contacts of transfected cells (Fig. 4, a′–c′). GFP-tagged PKP3ΔHR2 protein displays a localization very similar to full-length PKP3 (Fig. 4 d). The HR2 domain is the only conserved sequence stretch in the head domain of PKPs (Bonné et al., 1999). The intense accumulation in desmosomes of this mutated protein overlaps with endogenous DP (Fig. 4, e and f). Larger magnifications clearly display the punctate (co) localization along cell–cell contacts of transfected cells (Fig. 4, d′–f'′ and d′′–f'′′). In contrast, the GFP-tagged PKP3arm fragment localizes in the cell nucleus, whereas only a faint localization is observed at cell–cell contacts (Fig. 4 g); DP detection in the same cell field is shown in Fig. 4 h. The GFP-tagged PKP3head domain is often observed as discrete aggregates in the cytoplasm and localizes only weakly in cell–cell contacts (Fig. 4 i); control detection of DP in the same field is shown in Fig. 4 j. In MCF7/AZ cells, a similar distribution of the GFP-PKP3head domain is observed (Fig. 4 k); detection of endogenous PKP3 in the same field using mAb 23E3/4 is shown in Fig. 4 l. In MCF7/AZ cells, cell–cell contacts are often less pronounced compared with HCT8/E8 cells, and endogenous PKP3 was sometimes found to colocalize with GFP-PKP3head aggregates (Fig. 4, k and l). Finally, exogenous PKP3headΔHR2 displayed an intracellular localization pattern like PKP3head (unpublished data).


Defining desmosomal plakophilin-3 interactions.

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

Intracellular localization of exogenous GFP-tagged PKP3 and fragments thereof in HCT8/E8 and MCF7/AZ cells. In HCT8/E8 cells, full-length GFP-tagged PKP3 localizes predominantly in desmosomes and to a lesser extent in the cytoplasm (a). PKP3GFP and DP (b) overlap in desmosomes of transfected cells (c). The punctate localization of these proteins along cell–cell contacts is clear from the larger magnifications (a′–c′) of the fields indicated by arrowheads in pictures a–c. GFP-tagged PKP3ΔHR2 displays a similar intracellular localization as the full-length PKP3 (d), and colocalizes with DP (e) in desmosomes of transfected cells (f). The punctate localization of these proteins along cell–cell contacts is obvious from the larger magnifications (d′–f′) of the fields indicated by arrowheads in pictures d–f. As is clear from images d′′–f′′ (fields indicated by arrows in pictures d–f), GFP-tagged PKP3 overlaps only half of the DP signal if transfected cells contact untransfected cells. PKP3arm GFP accumulates to high levels in the cell nucleus, but almost not at all at cell–cell contacts (g); control immunodetection of DP in the same cell field (h). GFP-tagged PKP3head protein accumulates as discrete cytoplasmic aggregates, in addition to a more diffuse cytoplasmic localization and less intense accumulation at cell–cell contacts (i); control detection of DP in the same field (j). GFP-tagged PKP3head is observed in a similar pattern in MCF7/AZ cells (k); endogenous PKP3 is detected using antibody 23E3/4 (l). Arrowheads, occasional endogenous PKP3 localization in PKP3headGFP aggregates; arrows, colocalization of exogenous fusion protein and endogenous PKP3 at sites of cell–cell contact. The marked regions are enlarged in the insets. Bars: 10 μm (a–j) and 20 μm (k and l).
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fig4: Intracellular localization of exogenous GFP-tagged PKP3 and fragments thereof in HCT8/E8 and MCF7/AZ cells. In HCT8/E8 cells, full-length GFP-tagged PKP3 localizes predominantly in desmosomes and to a lesser extent in the cytoplasm (a). PKP3GFP and DP (b) overlap in desmosomes of transfected cells (c). The punctate localization of these proteins along cell–cell contacts is clear from the larger magnifications (a′–c′) of the fields indicated by arrowheads in pictures a–c. GFP-tagged PKP3ΔHR2 displays a similar intracellular localization as the full-length PKP3 (d), and colocalizes with DP (e) in desmosomes of transfected cells (f). The punctate localization of these proteins along cell–cell contacts is obvious from the larger magnifications (d′–f′) of the fields indicated by arrowheads in pictures d–f. As is clear from images d′′–f′′ (fields indicated by arrows in pictures d–f), GFP-tagged PKP3 overlaps only half of the DP signal if transfected cells contact untransfected cells. PKP3arm GFP accumulates to high levels in the cell nucleus, but almost not at all at cell–cell contacts (g); control immunodetection of DP in the same cell field (h). GFP-tagged PKP3head protein accumulates as discrete cytoplasmic aggregates, in addition to a more diffuse cytoplasmic localization and less intense accumulation at cell–cell contacts (i); control detection of DP in the same field (j). GFP-tagged PKP3head is observed in a similar pattern in MCF7/AZ cells (k); endogenous PKP3 is detected using antibody 23E3/4 (l). Arrowheads, occasional endogenous PKP3 localization in PKP3headGFP aggregates; arrows, colocalization of exogenous fusion protein and endogenous PKP3 at sites of cell–cell contact. The marked regions are enlarged in the insets. Bars: 10 μm (a–j) and 20 μm (k and l).
Mentions: To study the intracellular distribution of full-length PKP3 and fragments thereof, transfection of PKP3 expression constructs was performed in HCT8/E8, MCF7/AZ, COS1, and PtK2 cells, yielding consistent data. A schematic overview of the protein fragments encoded by the different constructs is shown in Fig. 3 . In HCT8/E8 cells, full-length human PKP3 tagged with GFP displays an intracellular distribution similar to endogenous PKP3 detected by mAbs, i.e., a clear-cut localization in desmosomes, weaker in the cytoplasm, and absent from the cell nucleus (Fig. 4 a). Exogenous PKP3 colocalizes with endogenous DP in desmosomes (Fig. 4, b and c). Larger magnifications emphasize the punctate (co)localization of these proteins along cell–cell contacts of transfected cells (Fig. 4, a′–c′). GFP-tagged PKP3ΔHR2 protein displays a localization very similar to full-length PKP3 (Fig. 4 d). The HR2 domain is the only conserved sequence stretch in the head domain of PKPs (Bonné et al., 1999). The intense accumulation in desmosomes of this mutated protein overlaps with endogenous DP (Fig. 4, e and f). Larger magnifications clearly display the punctate (co) localization along cell–cell contacts of transfected cells (Fig. 4, d′–f'′ and d′′–f'′′). In contrast, the GFP-tagged PKP3arm fragment localizes in the cell nucleus, whereas only a faint localization is observed at cell–cell contacts (Fig. 4 g); DP detection in the same cell field is shown in Fig. 4 h. The GFP-tagged PKP3head domain is often observed as discrete aggregates in the cytoplasm and localizes only weakly in cell–cell contacts (Fig. 4 i); control detection of DP in the same field is shown in Fig. 4 j. In MCF7/AZ cells, a similar distribution of the GFP-PKP3head domain is observed (Fig. 4 k); detection of endogenous PKP3 in the same field using mAb 23E3/4 is shown in Fig. 4 l. In MCF7/AZ cells, cell–cell contacts are often less pronounced compared with HCT8/E8 cells, and endogenous PKP3 was sometimes found to colocalize with GFP-PKP3head aggregates (Fig. 4, k and l). Finally, exogenous PKP3headΔHR2 displayed an intracellular localization pattern like PKP3head (unpublished data).

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