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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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Immunodetection of PKPs using different mouse mAbs. PKP3, detected with mAb 23E3/4 (a and b), 12B11F8 (c), or PKP2 (d) show a similar expression pattern along cell–cell contacts in methanol-fixed HaCaT (a) and HCT8/E8 (b–d) cells. Larger magnifications display the punctate localization of these proteins along cell–cell contacts (a′–d′). Bar, 10 μm. (e) Western blot detection of PKP3 (lane 1, mAb 12B11F8; lane 2, 23E3/4), PKP2 (lane 3), and PKP1 (lane 4) in HaCaT protein lysates. Equal amounts of protein were loaded in each lane.
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fig1: Immunodetection of PKPs using different mouse mAbs. PKP3, detected with mAb 23E3/4 (a and b), 12B11F8 (c), or PKP2 (d) show a similar expression pattern along cell–cell contacts in methanol-fixed HaCaT (a) and HCT8/E8 (b–d) cells. Larger magnifications display the punctate localization of these proteins along cell–cell contacts (a′–d′). Bar, 10 μm. (e) Western blot detection of PKP3 (lane 1, mAb 12B11F8; lane 2, 23E3/4), PKP2 (lane 3), and PKP1 (lane 4) in HaCaT protein lysates. Equal amounts of protein were loaded in each lane.

Mentions: In immunofluorescence detection experiments using the newly generated anti-PKP3 mAbs 23E3/4 and 12B11F8 raised against different PKP3-specific peptides, we detected PKP3 in a desmosomal punctate pattern along cell–cell contacts, besides weak immunopositivity in the cytoplasm. This observation was made in both methanol- and formaldehyde-fixed cells (exemplified in Fig. 1 , a–c). Control immunodetections of DP (unpublished data) or PKP2 (Fig. 1 d) revealed a similar pattern along cell–cell contacts. The punctate localization of PKP3 and PKP2 along cell–cell contacts is also obvious from the larger magnifications (Fig. 1, a′–d′). Western blot experiments showed that these novel mouse anti-PKP3 mAbs do not cross react with either PKP1 or PKP2 (Fig. 1 e). However, using these mAbs we could not confirm nuclear localization of PKP3 observed previously with a rabbit pAb (Bonné et al., 1999). Intriguingly, mAb 12B11F8 was raised against the same antigenic peptide as this rabbit pAb. Therefore, the reported nuclear PKP3 localization using the rabbit pAb may be caused by additional immunoreactivity, unrelated to PKP3 despite the fact that preincubation of the polyclonal antiserum with the antigenic peptide could block its activity (Bonné et al., 1999).


Defining desmosomal plakophilin-3 interactions.

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

Immunodetection of PKPs using different mouse mAbs. PKP3, detected with mAb 23E3/4 (a and b), 12B11F8 (c), or PKP2 (d) show a similar expression pattern along cell–cell contacts in methanol-fixed HaCaT (a) and HCT8/E8 (b–d) cells. Larger magnifications display the punctate localization of these proteins along cell–cell contacts (a′–d′). Bar, 10 μm. (e) Western blot detection of PKP3 (lane 1, mAb 12B11F8; lane 2, 23E3/4), PKP2 (lane 3), and PKP1 (lane 4) in HaCaT protein lysates. Equal amounts of protein were loaded in each lane.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172904&req=5

fig1: Immunodetection of PKPs using different mouse mAbs. PKP3, detected with mAb 23E3/4 (a and b), 12B11F8 (c), or PKP2 (d) show a similar expression pattern along cell–cell contacts in methanol-fixed HaCaT (a) and HCT8/E8 (b–d) cells. Larger magnifications display the punctate localization of these proteins along cell–cell contacts (a′–d′). Bar, 10 μm. (e) Western blot detection of PKP3 (lane 1, mAb 12B11F8; lane 2, 23E3/4), PKP2 (lane 3), and PKP1 (lane 4) in HaCaT protein lysates. Equal amounts of protein were loaded in each lane.
Mentions: In immunofluorescence detection experiments using the newly generated anti-PKP3 mAbs 23E3/4 and 12B11F8 raised against different PKP3-specific peptides, we detected PKP3 in a desmosomal punctate pattern along cell–cell contacts, besides weak immunopositivity in the cytoplasm. This observation was made in both methanol- and formaldehyde-fixed cells (exemplified in Fig. 1 , a–c). Control immunodetections of DP (unpublished data) or PKP2 (Fig. 1 d) revealed a similar pattern along cell–cell contacts. The punctate localization of PKP3 and PKP2 along cell–cell contacts is also obvious from the larger magnifications (Fig. 1, a′–d′). Western blot experiments showed that these novel mouse anti-PKP3 mAbs do not cross react with either PKP1 or PKP2 (Fig. 1 e). However, using these mAbs we could not confirm nuclear localization of PKP3 observed previously with a rabbit pAb (Bonné et al., 1999). Intriguingly, mAb 12B11F8 was raised against the same antigenic peptide as this rabbit pAb. Therefore, the reported nuclear PKP3 localization using the rabbit pAb may be caused by additional immunoreactivity, unrelated to PKP3 despite the fact that preincubation of the polyclonal antiserum with the antigenic peptide could block its activity (Bonné et al., 1999).

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