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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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Desmosome model showing interactions between selected molecular components of simple and stratified epithelia (modified after Nollet et al., 2000). (a) Some representative desmosomal proteins are depicted. CBS, catenin-binding segment; CK, cytokeratin; EC, ectodomain module; IA, intracellular anchor domain; MPED, membrane-proximal extracellular domain; N, amino-terminal domain; PL, proline-rich linker; PM, plasma membrane; RUD, repeat unit domain; TD, terminal domain. In the present work, the combination (PL-RUDs-TD) was designated Dsg domain (Hatzfeld et al., 2000). (b) The localizations in simple epithelia of PKP1, PKP2, and PKP3 as compared with Pg are mainly based on observations made by others (Mertens et al., 1996; North et al., 1999; Schmidt et al., 1999). DP occurs as two splice variants, DPI and the shorter DPII that is expressed in epithelia, but not in heart (Kowalczyk et al., 1999b). The stoichiometry of the interactions between desmosomal plaque molecules is unclear. For the Pg–Dsg1 interaction, ratios >1:1 have been reported (Bannon et al., 2001). It is also unclear which and how many proteins can bind at the same time to a single PKP protein. Here, we have shown that at least the PKP3 head domain contains two DP interaction sites. (c) The location and multimolecular interactions of PKP1 in stratified epithelia are adapted from a model proposed by Kowalczyk et al. (1999a). According to immunoelectron localization studies, the carboxy-termini of DPI and DPII are localized at the same distance from the cell membrane that is not reflected here. In epidermis, PKP1, Dsc1, and Dsg1 are enriched in the superficial layers, whereas Dsg3, Dsc3, and PKP2 are concentrated in the basal layers. PKP3 is expressed throughout all living cell layers of the epidermis (Fig. 2).
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fig11: Desmosome model showing interactions between selected molecular components of simple and stratified epithelia (modified after Nollet et al., 2000). (a) Some representative desmosomal proteins are depicted. CBS, catenin-binding segment; CK, cytokeratin; EC, ectodomain module; IA, intracellular anchor domain; MPED, membrane-proximal extracellular domain; N, amino-terminal domain; PL, proline-rich linker; PM, plasma membrane; RUD, repeat unit domain; TD, terminal domain. In the present work, the combination (PL-RUDs-TD) was designated Dsg domain (Hatzfeld et al., 2000). (b) The localizations in simple epithelia of PKP1, PKP2, and PKP3 as compared with Pg are mainly based on observations made by others (Mertens et al., 1996; North et al., 1999; Schmidt et al., 1999). DP occurs as two splice variants, DPI and the shorter DPII that is expressed in epithelia, but not in heart (Kowalczyk et al., 1999b). The stoichiometry of the interactions between desmosomal plaque molecules is unclear. For the Pg–Dsg1 interaction, ratios >1:1 have been reported (Bannon et al., 2001). It is also unclear which and how many proteins can bind at the same time to a single PKP protein. Here, we have shown that at least the PKP3 head domain contains two DP interaction sites. (c) The location and multimolecular interactions of PKP1 in stratified epithelia are adapted from a model proposed by Kowalczyk et al. (1999a). According to immunoelectron localization studies, the carboxy-termini of DPI and DPII are localized at the same distance from the cell membrane that is not reflected here. In epidermis, PKP1, Dsc1, and Dsg1 are enriched in the superficial layers, whereas Dsg3, Dsc3, and PKP2 are concentrated in the basal layers. PKP3 is expressed throughout all living cell layers of the epidermis (Fig. 2).

Mentions: Direct interactions between PKP3 and desmosomal cadherins were investigated by yeast two-hybrid analysis. The PKP3 protein fragments used are depicted in Fig. 3. Results are shown in Fig. 7 and summarized in Table I. Full-length PKP3 interacts with all three Dsgs (Fig. 7 a). Using deletion constructs, the Dsg1-binding site was confined to the head domain of PKP3, whereas the binding site of Dsg2 and Dsg3 apparently encompass (parts of) both the head and arm domain of PKP3 (Table I). Deletion of the HR2 domain had no effect on PKP3 binding with Dsgs (Table I). Also, binding of PKP3 with Dsc1a, Dsc2a, and Dsc3a was observed (Fig. 7 a). Interaction of full-length PKP3 with Dsc1b and Dsc2b remained unclear (colonies grew slower and turned only light blue), whereas the PKP3 interaction with Dsc3b was seemingly weaker than its interactions with Dsc-a forms. Nonetheless, the PKP3–Dsc3a interaction observed was not completely convincing because all PKP3 deletion constructs cotransformed with Dsc3a interacted with this desmosomal cadherin, for which we have no explanation (Table I). Both arm and head domains of PKP3 were necessary for interaction with Dscs, but deletion of the HR2 domain did not influence the interaction with Dscs (Table I). Using deletion constructs of the Dsg1 cytoplasmic domain in the bait plasmid pGADT7, we found two separate PKP3 interaction sites. Although the intracellular anchoring domain (IA) and catenin-binding segment (CBS) domains alone did not interact with PKP3, combination of both did (Fig. 7 b). On the other hand, interaction was also observed with the Dsg domain of Dsg1, i.e., the desmoglein-specific domain composed of a proline-rich linker segment, a repeat unit domain, and a terminal domain (Hatzfeld et al., 2000; see Fig. 11), implying the presence of two physically separable PKP3 interaction sites in the Dsg1 cytoplasmic domain (Fig. 7 b). The presence of two independent interaction sites was also observed with constructs of Dsg2 in pGADT7, as we found that both aa domains 583–721 and 882–1069 interact with full-length PKP3 (Fig. 7 b). As controls, all PKP3 constructs were cotransformed with several constructs including one encoding the mouse E-cadherin cytoplasmic tail, whereas desmosomal cadherin constructs were cotransformed with human p120ctn isoform 3AC. None of these cotransformed yeast clones grew on interaction-selective medium, whereas E-cadherin interacted with p120ctn (Fig. 7 a; Table I). Absence of growth of yeast clones was also observed when PKP3 bait plasmids were cotransformed with empty prey vectors, and when empty bait plasmids were cotransformed with prey constructs encoding desmosomal cadherins (Table I).


Defining desmosomal plakophilin-3 interactions.

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

Desmosome model showing interactions between selected molecular components of simple and stratified epithelia (modified after Nollet et al., 2000). (a) Some representative desmosomal proteins are depicted. CBS, catenin-binding segment; CK, cytokeratin; EC, ectodomain module; IA, intracellular anchor domain; MPED, membrane-proximal extracellular domain; N, amino-terminal domain; PL, proline-rich linker; PM, plasma membrane; RUD, repeat unit domain; TD, terminal domain. In the present work, the combination (PL-RUDs-TD) was designated Dsg domain (Hatzfeld et al., 2000). (b) The localizations in simple epithelia of PKP1, PKP2, and PKP3 as compared with Pg are mainly based on observations made by others (Mertens et al., 1996; North et al., 1999; Schmidt et al., 1999). DP occurs as two splice variants, DPI and the shorter DPII that is expressed in epithelia, but not in heart (Kowalczyk et al., 1999b). The stoichiometry of the interactions between desmosomal plaque molecules is unclear. For the Pg–Dsg1 interaction, ratios >1:1 have been reported (Bannon et al., 2001). It is also unclear which and how many proteins can bind at the same time to a single PKP protein. Here, we have shown that at least the PKP3 head domain contains two DP interaction sites. (c) The location and multimolecular interactions of PKP1 in stratified epithelia are adapted from a model proposed by Kowalczyk et al. (1999a). According to immunoelectron localization studies, the carboxy-termini of DPI and DPII are localized at the same distance from the cell membrane that is not reflected here. In epidermis, PKP1, Dsc1, and Dsg1 are enriched in the superficial layers, whereas Dsg3, Dsc3, and PKP2 are concentrated in the basal layers. PKP3 is expressed throughout all living cell layers of the epidermis (Fig. 2).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172904&req=5

fig11: Desmosome model showing interactions between selected molecular components of simple and stratified epithelia (modified after Nollet et al., 2000). (a) Some representative desmosomal proteins are depicted. CBS, catenin-binding segment; CK, cytokeratin; EC, ectodomain module; IA, intracellular anchor domain; MPED, membrane-proximal extracellular domain; N, amino-terminal domain; PL, proline-rich linker; PM, plasma membrane; RUD, repeat unit domain; TD, terminal domain. In the present work, the combination (PL-RUDs-TD) was designated Dsg domain (Hatzfeld et al., 2000). (b) The localizations in simple epithelia of PKP1, PKP2, and PKP3 as compared with Pg are mainly based on observations made by others (Mertens et al., 1996; North et al., 1999; Schmidt et al., 1999). DP occurs as two splice variants, DPI and the shorter DPII that is expressed in epithelia, but not in heart (Kowalczyk et al., 1999b). The stoichiometry of the interactions between desmosomal plaque molecules is unclear. For the Pg–Dsg1 interaction, ratios >1:1 have been reported (Bannon et al., 2001). It is also unclear which and how many proteins can bind at the same time to a single PKP protein. Here, we have shown that at least the PKP3 head domain contains two DP interaction sites. (c) The location and multimolecular interactions of PKP1 in stratified epithelia are adapted from a model proposed by Kowalczyk et al. (1999a). According to immunoelectron localization studies, the carboxy-termini of DPI and DPII are localized at the same distance from the cell membrane that is not reflected here. In epidermis, PKP1, Dsc1, and Dsg1 are enriched in the superficial layers, whereas Dsg3, Dsc3, and PKP2 are concentrated in the basal layers. PKP3 is expressed throughout all living cell layers of the epidermis (Fig. 2).
Mentions: Direct interactions between PKP3 and desmosomal cadherins were investigated by yeast two-hybrid analysis. The PKP3 protein fragments used are depicted in Fig. 3. Results are shown in Fig. 7 and summarized in Table I. Full-length PKP3 interacts with all three Dsgs (Fig. 7 a). Using deletion constructs, the Dsg1-binding site was confined to the head domain of PKP3, whereas the binding site of Dsg2 and Dsg3 apparently encompass (parts of) both the head and arm domain of PKP3 (Table I). Deletion of the HR2 domain had no effect on PKP3 binding with Dsgs (Table I). Also, binding of PKP3 with Dsc1a, Dsc2a, and Dsc3a was observed (Fig. 7 a). Interaction of full-length PKP3 with Dsc1b and Dsc2b remained unclear (colonies grew slower and turned only light blue), whereas the PKP3 interaction with Dsc3b was seemingly weaker than its interactions with Dsc-a forms. Nonetheless, the PKP3–Dsc3a interaction observed was not completely convincing because all PKP3 deletion constructs cotransformed with Dsc3a interacted with this desmosomal cadherin, for which we have no explanation (Table I). Both arm and head domains of PKP3 were necessary for interaction with Dscs, but deletion of the HR2 domain did not influence the interaction with Dscs (Table I). Using deletion constructs of the Dsg1 cytoplasmic domain in the bait plasmid pGADT7, we found two separate PKP3 interaction sites. Although the intracellular anchoring domain (IA) and catenin-binding segment (CBS) domains alone did not interact with PKP3, combination of both did (Fig. 7 b). On the other hand, interaction was also observed with the Dsg domain of Dsg1, i.e., the desmoglein-specific domain composed of a proline-rich linker segment, a repeat unit domain, and a terminal domain (Hatzfeld et al., 2000; see Fig. 11), implying the presence of two physically separable PKP3 interaction sites in the Dsg1 cytoplasmic domain (Fig. 7 b). The presence of two independent interaction sites was also observed with constructs of Dsg2 in pGADT7, as we found that both aa domains 583–721 and 882–1069 interact with full-length PKP3 (Fig. 7 b). As controls, all PKP3 constructs were cotransformed with several constructs including one encoding the mouse E-cadherin cytoplasmic tail, whereas desmosomal cadherin constructs were cotransformed with human p120ctn isoform 3AC. None of these cotransformed yeast clones grew on interaction-selective medium, whereas E-cadherin interacted with p120ctn (Fig. 7 a; Table I). Absence of growth of yeast clones was also observed when PKP3 bait plasmids were cotransformed with empty prey vectors, and when empty bait plasmids were cotransformed with prey constructs encoding desmosomal cadherins (Table I).

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