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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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Yeast two-hybrid results using PKP3 and fragments thereof as baits. Two colonies are shown for each cotransformation, including negative controls. SD-LWHA is the medium indicative of protein interactions, as it is supplemented with Xα-gal, whereas SD-LW is indicative of successful cotransformation of bait and prey plasmids. (a) Full-length PKP3 interacts with Dsg1, Dsg2, Dsg3, Dsc1a, Dsc2a, and Dsc3a. Interaction with Dsc1b and Dsc2b is unclear, whereas PKP3 interaction with Dsc3b seems to be less strong than its interaction with Dsc-a proteins. No interaction is observed between desmosomal cadherins and p120ctn. Pg also interacts with PKP3 but not with protocadherin-β15 (pcdβ15). No interaction is observed between PKP3 and E-cadherin (E-cad), whereas E-cadherin and p120ctn clearly interact. (b) Defining the PKP3 interaction sites within the Dsg1 (aa 519–1000) and Dsg2 (aa 583–1069) cytoplasmic domains. Non-interacting Dsg1 protein fragments are indicated by broken lines. Separate IA and CBS domains do not interact with PKP3, and neither does a construct containing aa 593–789 of Dsg1 (i.e., the CBS domain plus part of the Dsg domain). Together, the IA and CBS domains interact with PKP3. The Dsg domain alone is also sufficient for interaction with PKP3. As such, two independent PKP3-interacting domains can be identified in the Dsg1 cytoplasmic domain. In the case of Dsg2, again two separate PKP3 interaction sites can be detected, i.e., aa 583–721 and aa 882–1069. IA, intracellular anchor; CBS, catenin-binding segment; Dsg, desmoglein-specific domain; C, carboxy terminus. (c) Full-length PKP3 and PKP3head, but not PKP3arm, interact with both DPNTP and DPNTPmut. PKP3ΔHR2 and PKP3headΔHR2 bind to DPNTP, but not to DPNTPmut. Only PKP3arm does not interact with CK18. None of the PKP3 constructs interacted with E-cadherin in the yeast two-hybrid system.
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fig7: Yeast two-hybrid results using PKP3 and fragments thereof as baits. Two colonies are shown for each cotransformation, including negative controls. SD-LWHA is the medium indicative of protein interactions, as it is supplemented with Xα-gal, whereas SD-LW is indicative of successful cotransformation of bait and prey plasmids. (a) Full-length PKP3 interacts with Dsg1, Dsg2, Dsg3, Dsc1a, Dsc2a, and Dsc3a. Interaction with Dsc1b and Dsc2b is unclear, whereas PKP3 interaction with Dsc3b seems to be less strong than its interaction with Dsc-a proteins. No interaction is observed between desmosomal cadherins and p120ctn. Pg also interacts with PKP3 but not with protocadherin-β15 (pcdβ15). No interaction is observed between PKP3 and E-cadherin (E-cad), whereas E-cadherin and p120ctn clearly interact. (b) Defining the PKP3 interaction sites within the Dsg1 (aa 519–1000) and Dsg2 (aa 583–1069) cytoplasmic domains. Non-interacting Dsg1 protein fragments are indicated by broken lines. Separate IA and CBS domains do not interact with PKP3, and neither does a construct containing aa 593–789 of Dsg1 (i.e., the CBS domain plus part of the Dsg domain). Together, the IA and CBS domains interact with PKP3. The Dsg domain alone is also sufficient for interaction with PKP3. As such, two independent PKP3-interacting domains can be identified in the Dsg1 cytoplasmic domain. In the case of Dsg2, again two separate PKP3 interaction sites can be detected, i.e., aa 583–721 and aa 882–1069. IA, intracellular anchor; CBS, catenin-binding segment; Dsg, desmoglein-specific domain; C, carboxy terminus. (c) Full-length PKP3 and PKP3head, but not PKP3arm, interact with both DPNTP and DPNTPmut. PKP3ΔHR2 and PKP3headΔHR2 bind to DPNTP, but not to DPNTPmut. Only PKP3arm does not interact with CK18. None of the PKP3 constructs interacted with E-cadherin in the yeast two-hybrid system.

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)

Yeast two-hybrid results using PKP3 and fragments thereof as baits. Two colonies are shown for each cotransformation, including negative controls. SD-LWHA is the medium indicative of protein interactions, as it is supplemented with Xα-gal, whereas SD-LW is indicative of successful cotransformation of bait and prey plasmids. (a) Full-length PKP3 interacts with Dsg1, Dsg2, Dsg3, Dsc1a, Dsc2a, and Dsc3a. Interaction with Dsc1b and Dsc2b is unclear, whereas PKP3 interaction with Dsc3b seems to be less strong than its interaction with Dsc-a proteins. No interaction is observed between desmosomal cadherins and p120ctn. Pg also interacts with PKP3 but not with protocadherin-β15 (pcdβ15). No interaction is observed between PKP3 and E-cadherin (E-cad), whereas E-cadherin and p120ctn clearly interact. (b) Defining the PKP3 interaction sites within the Dsg1 (aa 519–1000) and Dsg2 (aa 583–1069) cytoplasmic domains. Non-interacting Dsg1 protein fragments are indicated by broken lines. Separate IA and CBS domains do not interact with PKP3, and neither does a construct containing aa 593–789 of Dsg1 (i.e., the CBS domain plus part of the Dsg domain). Together, the IA and CBS domains interact with PKP3. The Dsg domain alone is also sufficient for interaction with PKP3. As such, two independent PKP3-interacting domains can be identified in the Dsg1 cytoplasmic domain. In the case of Dsg2, again two separate PKP3 interaction sites can be detected, i.e., aa 583–721 and aa 882–1069. IA, intracellular anchor; CBS, catenin-binding segment; Dsg, desmoglein-specific domain; C, carboxy terminus. (c) Full-length PKP3 and PKP3head, but not PKP3arm, interact with both DPNTP and DPNTPmut. PKP3ΔHR2 and PKP3headΔHR2 bind to DPNTP, but not to DPNTPmut. Only PKP3arm does not interact with CK18. None of the PKP3 constructs interacted with E-cadherin in the yeast two-hybrid system.
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Related In: Results  -  Collection

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fig7: Yeast two-hybrid results using PKP3 and fragments thereof as baits. Two colonies are shown for each cotransformation, including negative controls. SD-LWHA is the medium indicative of protein interactions, as it is supplemented with Xα-gal, whereas SD-LW is indicative of successful cotransformation of bait and prey plasmids. (a) Full-length PKP3 interacts with Dsg1, Dsg2, Dsg3, Dsc1a, Dsc2a, and Dsc3a. Interaction with Dsc1b and Dsc2b is unclear, whereas PKP3 interaction with Dsc3b seems to be less strong than its interaction with Dsc-a proteins. No interaction is observed between desmosomal cadherins and p120ctn. Pg also interacts with PKP3 but not with protocadherin-β15 (pcdβ15). No interaction is observed between PKP3 and E-cadherin (E-cad), whereas E-cadherin and p120ctn clearly interact. (b) Defining the PKP3 interaction sites within the Dsg1 (aa 519–1000) and Dsg2 (aa 583–1069) cytoplasmic domains. Non-interacting Dsg1 protein fragments are indicated by broken lines. Separate IA and CBS domains do not interact with PKP3, and neither does a construct containing aa 593–789 of Dsg1 (i.e., the CBS domain plus part of the Dsg domain). Together, the IA and CBS domains interact with PKP3. The Dsg domain alone is also sufficient for interaction with PKP3. As such, two independent PKP3-interacting domains can be identified in the Dsg1 cytoplasmic domain. In the case of Dsg2, again two separate PKP3 interaction sites can be detected, i.e., aa 583–721 and aa 882–1069. IA, intracellular anchor; CBS, catenin-binding segment; Dsg, desmoglein-specific domain; C, carboxy terminus. (c) Full-length PKP3 and PKP3head, but not PKP3arm, interact with both DPNTP and DPNTPmut. PKP3ΔHR2 and PKP3headΔHR2 bind to DPNTP, but not to DPNTPmut. Only PKP3arm does not interact with CK18. None of the PKP3 constructs interacted with E-cadherin in the yeast two-hybrid system.
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