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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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CoIP experiments using lysates of HEK293T cells cotransfected with plasmids of interest. A plasmid encoding full-length PKP3 (p1744) was cotransfected with plasmids encoding Dsg1-myc, Dsg2-myc, Dsg3-myc, Dsc3a-myc, Dsc3b-myc, Dsc3bΔcyto1-myc, Dsc3bΔcyto2-myc, DP-myc, or Pg-myc fusion proteins. (a) Using anti-PKP3 mAb 23E3/4, myc-tagged Dsg1, Dsg2, Dsg3, Dsc3b, DP, and Pg proteins (arrows) were coimmunoprecipitated with PKP3 (+ lanes). The nature of the Dsc3b doublet is unclear, but might represent incompletely processed protein in addition to mature protein. In the negative control lanes (lysates incubated with protein G Sepharose in the absence of mAb 23E3/4), no signal could be detected (− lanes). Incompletely reduced primary antibody was often detected (star). Identical exposure times were used for each set of +/− lanes. (b) Control Western blot detection of total cell lysates: Dsg1 (165 kD), Dsg2 (160 kD), Dsg3 (135 kD), Dsc3b (99 kD), DP (250 kD, arrow), and Pg (82 kD) in the left panel; PKP3 (87 kD) in the right panel. Mol wt markers (in kD) are indicated. (c) Using anti-PKP3 mAb 23E3/4, myc-tagged Dsc3b, two COOH-terminally truncated derivatives of Dsc3b and Dsc3a were coimmunoprecipitated with PKP3 (+ lanes in left panel). In the negative control lanes (lysates incubated with protein G Sepharose in the absence of mAb 23E3/4), no signal could be detected (− lanes in left panel). Expression of each of these fusion proteins was detected by Western blotting (right), using anti-Myc and anti-PKP3 antibodies. (d) Schematic representation of the cytoplasmic domains of mouse (Mm) Dsc3a, Dsc3b, and Dsc3b truncation mutants used in the CoIP experiments of (C). Dashed lines indicate isoform-specific domains. The fragment containing aa 578–696 is shared by Dsc3a and Dsc3b. Dsc3bΔcyto1 and Dsc3bΔcyto2 encompass, respectively, 36 and 77 membrane-proximal aa of both Dsc3a and -b.
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fig8: CoIP experiments using lysates of HEK293T cells cotransfected with plasmids of interest. A plasmid encoding full-length PKP3 (p1744) was cotransfected with plasmids encoding Dsg1-myc, Dsg2-myc, Dsg3-myc, Dsc3a-myc, Dsc3b-myc, Dsc3bΔcyto1-myc, Dsc3bΔcyto2-myc, DP-myc, or Pg-myc fusion proteins. (a) Using anti-PKP3 mAb 23E3/4, myc-tagged Dsg1, Dsg2, Dsg3, Dsc3b, DP, and Pg proteins (arrows) were coimmunoprecipitated with PKP3 (+ lanes). The nature of the Dsc3b doublet is unclear, but might represent incompletely processed protein in addition to mature protein. In the negative control lanes (lysates incubated with protein G Sepharose in the absence of mAb 23E3/4), no signal could be detected (− lanes). Incompletely reduced primary antibody was often detected (star). Identical exposure times were used for each set of +/− lanes. (b) Control Western blot detection of total cell lysates: Dsg1 (165 kD), Dsg2 (160 kD), Dsg3 (135 kD), Dsc3b (99 kD), DP (250 kD, arrow), and Pg (82 kD) in the left panel; PKP3 (87 kD) in the right panel. Mol wt markers (in kD) are indicated. (c) Using anti-PKP3 mAb 23E3/4, myc-tagged Dsc3b, two COOH-terminally truncated derivatives of Dsc3b and Dsc3a were coimmunoprecipitated with PKP3 (+ lanes in left panel). In the negative control lanes (lysates incubated with protein G Sepharose in the absence of mAb 23E3/4), no signal could be detected (− lanes in left panel). Expression of each of these fusion proteins was detected by Western blotting (right), using anti-Myc and anti-PKP3 antibodies. (d) Schematic representation of the cytoplasmic domains of mouse (Mm) Dsc3a, Dsc3b, and Dsc3b truncation mutants used in the CoIP experiments of (C). Dashed lines indicate isoform-specific domains. The fragment containing aa 578–696 is shared by Dsc3a and Dsc3b. Dsc3bΔcyto1 and Dsc3bΔcyto2 encompass, respectively, 36 and 77 membrane-proximal aa of both Dsc3a and -b.

Mentions: To confirm the interactions observed in the yeast two-hybrid system, coimmunoprecipitation (CoIP) experiments were performed upon cotransfection of the relevant expression plasmids in HEK293T fibroblasts. These cells have no functional desmosomes and their endogenous protein levels of desmosomal components, such as Dsg2, PKP3, DP, and Pg, are very low or nil (unpublished data). We noticed that the full-length PKP3 protein sticks nonspecifically to protein G Sepharose beads, and because more stringent washings did not significantly reduce this binding, CoIP experiments were performed in one direction only. The PKP3 protein was immunoprecipitated using mAb 23E3/4, whereas all other proteins were myc-tagged. Incompletely reduced primary mouse antibody was sometimes detected in CoIP samples (Fig. 8 a, star). Full-length PKP3 protein precipitates the desmosomal cadherins Dsg1, Dsg2, Dsg3, Dsc3b (Fig. 8 a), and Dsc3a (Fig. 8 c). In parallel, lysates were incubated with protein G Sepharose beads only, and no signal could be observed in these samples (Fig. 8, a and c). Using Dsc3b deletion constructs (depicted in Fig. 8 d), the PKP3 interaction site was confined to the membrane-proximal domain of Dsc3b (Fig. 8 c, left). Control detection of proteins in total lysates is shown in Fig. 8 (b and c). In a similar experiment, we tried to CoIP Dsc1a and Dsc2a with full-length PKP3. Unfortunately, no detectable levels of Dsc protein could be observed in cell lysates of appropriately cotransfected HEK293T cells (unpublished data). Finally, we found that full-length PKP3 immunoprecipitates the desmosomal plaque proteins DP and Pg (Fig. 8 a). Again, no signal is observed in the negative control lanes (Fig. 8 a). As such, these results confirm our data obtained using the yeast two-hybrid system.


Defining desmosomal plakophilin-3 interactions.

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

CoIP experiments using lysates of HEK293T cells cotransfected with plasmids of interest. A plasmid encoding full-length PKP3 (p1744) was cotransfected with plasmids encoding Dsg1-myc, Dsg2-myc, Dsg3-myc, Dsc3a-myc, Dsc3b-myc, Dsc3bΔcyto1-myc, Dsc3bΔcyto2-myc, DP-myc, or Pg-myc fusion proteins. (a) Using anti-PKP3 mAb 23E3/4, myc-tagged Dsg1, Dsg2, Dsg3, Dsc3b, DP, and Pg proteins (arrows) were coimmunoprecipitated with PKP3 (+ lanes). The nature of the Dsc3b doublet is unclear, but might represent incompletely processed protein in addition to mature protein. In the negative control lanes (lysates incubated with protein G Sepharose in the absence of mAb 23E3/4), no signal could be detected (− lanes). Incompletely reduced primary antibody was often detected (star). Identical exposure times were used for each set of +/− lanes. (b) Control Western blot detection of total cell lysates: Dsg1 (165 kD), Dsg2 (160 kD), Dsg3 (135 kD), Dsc3b (99 kD), DP (250 kD, arrow), and Pg (82 kD) in the left panel; PKP3 (87 kD) in the right panel. Mol wt markers (in kD) are indicated. (c) Using anti-PKP3 mAb 23E3/4, myc-tagged Dsc3b, two COOH-terminally truncated derivatives of Dsc3b and Dsc3a were coimmunoprecipitated with PKP3 (+ lanes in left panel). In the negative control lanes (lysates incubated with protein G Sepharose in the absence of mAb 23E3/4), no signal could be detected (− lanes in left panel). Expression of each of these fusion proteins was detected by Western blotting (right), using anti-Myc and anti-PKP3 antibodies. (d) Schematic representation of the cytoplasmic domains of mouse (Mm) Dsc3a, Dsc3b, and Dsc3b truncation mutants used in the CoIP experiments of (C). Dashed lines indicate isoform-specific domains. The fragment containing aa 578–696 is shared by Dsc3a and Dsc3b. Dsc3bΔcyto1 and Dsc3bΔcyto2 encompass, respectively, 36 and 77 membrane-proximal aa of both Dsc3a and -b.
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Related In: Results  -  Collection

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fig8: CoIP experiments using lysates of HEK293T cells cotransfected with plasmids of interest. A plasmid encoding full-length PKP3 (p1744) was cotransfected with plasmids encoding Dsg1-myc, Dsg2-myc, Dsg3-myc, Dsc3a-myc, Dsc3b-myc, Dsc3bΔcyto1-myc, Dsc3bΔcyto2-myc, DP-myc, or Pg-myc fusion proteins. (a) Using anti-PKP3 mAb 23E3/4, myc-tagged Dsg1, Dsg2, Dsg3, Dsc3b, DP, and Pg proteins (arrows) were coimmunoprecipitated with PKP3 (+ lanes). The nature of the Dsc3b doublet is unclear, but might represent incompletely processed protein in addition to mature protein. In the negative control lanes (lysates incubated with protein G Sepharose in the absence of mAb 23E3/4), no signal could be detected (− lanes). Incompletely reduced primary antibody was often detected (star). Identical exposure times were used for each set of +/− lanes. (b) Control Western blot detection of total cell lysates: Dsg1 (165 kD), Dsg2 (160 kD), Dsg3 (135 kD), Dsc3b (99 kD), DP (250 kD, arrow), and Pg (82 kD) in the left panel; PKP3 (87 kD) in the right panel. Mol wt markers (in kD) are indicated. (c) Using anti-PKP3 mAb 23E3/4, myc-tagged Dsc3b, two COOH-terminally truncated derivatives of Dsc3b and Dsc3a were coimmunoprecipitated with PKP3 (+ lanes in left panel). In the negative control lanes (lysates incubated with protein G Sepharose in the absence of mAb 23E3/4), no signal could be detected (− lanes in left panel). Expression of each of these fusion proteins was detected by Western blotting (right), using anti-Myc and anti-PKP3 antibodies. (d) Schematic representation of the cytoplasmic domains of mouse (Mm) Dsc3a, Dsc3b, and Dsc3b truncation mutants used in the CoIP experiments of (C). Dashed lines indicate isoform-specific domains. The fragment containing aa 578–696 is shared by Dsc3a and Dsc3b. Dsc3bΔcyto1 and Dsc3bΔcyto2 encompass, respectively, 36 and 77 membrane-proximal aa of both Dsc3a and -b.
Mentions: To confirm the interactions observed in the yeast two-hybrid system, coimmunoprecipitation (CoIP) experiments were performed upon cotransfection of the relevant expression plasmids in HEK293T fibroblasts. These cells have no functional desmosomes and their endogenous protein levels of desmosomal components, such as Dsg2, PKP3, DP, and Pg, are very low or nil (unpublished data). We noticed that the full-length PKP3 protein sticks nonspecifically to protein G Sepharose beads, and because more stringent washings did not significantly reduce this binding, CoIP experiments were performed in one direction only. The PKP3 protein was immunoprecipitated using mAb 23E3/4, whereas all other proteins were myc-tagged. Incompletely reduced primary mouse antibody was sometimes detected in CoIP samples (Fig. 8 a, star). Full-length PKP3 protein precipitates the desmosomal cadherins Dsg1, Dsg2, Dsg3, Dsc3b (Fig. 8 a), and Dsc3a (Fig. 8 c). In parallel, lysates were incubated with protein G Sepharose beads only, and no signal could be observed in these samples (Fig. 8, a and c). Using Dsc3b deletion constructs (depicted in Fig. 8 d), the PKP3 interaction site was confined to the membrane-proximal domain of Dsc3b (Fig. 8 c, left). Control detection of proteins in total lysates is shown in Fig. 8 (b and c). In a similar experiment, we tried to CoIP Dsc1a and Dsc2a with full-length PKP3. Unfortunately, no detectable levels of Dsc protein could be observed in cell lysates of appropriately cotransfected HEK293T cells (unpublished data). Finally, we found that full-length PKP3 immunoprecipitates the desmosomal plaque proteins DP and Pg (Fig. 8 a). Again, no signal is observed in the negative control lanes (Fig. 8 a). As such, these results confirm our data obtained using the yeast two-hybrid system.

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