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Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.

Todorovic V, Koetsier JL, Godsel LM, Green KJ - Mol. Biol. Cell (2014)

Bottom Line: Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly.We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing.These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.

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Pkp3-dependent Rap1 activation is required for normal DP border localization. (A) Immunofluorescence staining showing the effects of EPAC activator (8-CPT-2Me-cAMP) treatment on DP border localization in control and Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Average DP border intensity for experiments in A. Bars represent mean ± SEM. ns, p > 0.05; ***p < 0.001 (ANOVA, Bonferroni). (C) Western blot showing the levels of GTP-bound (active) Rap1 in NHEK cells treated as indicated. Active Rap1 was pulled down using GST-RalGDS. Rap1 pull-down bands were normalized to total Rap1 using ImageJ. Ratio between activated Rap1 in control and Pkp3 KD cells shows a lack of increase in active Rap1 in Pkp3 KD cells upon calcium switch at 5 and 60 min. (D) Western blot showing the ectopic expression of GFP-tagged Rap1a in SCC9 cells compared with GFP-only control plasmid. nt, no transfection. (E) Immunofluorescence staining of DP showing the recovery of DP at the borders of Rap1-GFP– transfected Pkp3 KD cells. Note the lack of recovery in GFP-only cells. GFP and Rap1-GFP transfection efficiency is shown in the insets marked with white rectangles. Stars represent the cells that are GFP positive in the corresponding inset. Scale bar, 20 μm. (F) Average pixel intensities of DP at the borders of the cells in E. Bars represent mean ± SEM. ns, p >0.05, ***p < 0.001 (ANOVA, Bonferroni).
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Figure 6: Pkp3-dependent Rap1 activation is required for normal DP border localization. (A) Immunofluorescence staining showing the effects of EPAC activator (8-CPT-2Me-cAMP) treatment on DP border localization in control and Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Average DP border intensity for experiments in A. Bars represent mean ± SEM. ns, p > 0.05; ***p < 0.001 (ANOVA, Bonferroni). (C) Western blot showing the levels of GTP-bound (active) Rap1 in NHEK cells treated as indicated. Active Rap1 was pulled down using GST-RalGDS. Rap1 pull-down bands were normalized to total Rap1 using ImageJ. Ratio between activated Rap1 in control and Pkp3 KD cells shows a lack of increase in active Rap1 in Pkp3 KD cells upon calcium switch at 5 and 60 min. (D) Western blot showing the ectopic expression of GFP-tagged Rap1a in SCC9 cells compared with GFP-only control plasmid. nt, no transfection. (E) Immunofluorescence staining of DP showing the recovery of DP at the borders of Rap1-GFP– transfected Pkp3 KD cells. Note the lack of recovery in GFP-only cells. GFP and Rap1-GFP transfection efficiency is shown in the insets marked with white rectangles. Stars represent the cells that are GFP positive in the corresponding inset. Scale bar, 20 μm. (F) Average pixel intensities of DP at the borders of the cells in E. Bars represent mean ± SEM. ns, p >0.05, ***p < 0.001 (ANOVA, Bonferroni).

Mentions: The secondary messenger cAMP is responsible for regulating two major signaling axes: protein kinase A (PKA) activation and activation of the Rap1 pathway through the guanine exchange factor (GEF) exchange protein directly activated by cAMP (EPAC; Kopperud et al., 2003; Cheng et al., 2008). Despite our early findings that FSK treatment leads to DP phosphorylation and enhances its accumulation at cell–cell interfaces (Stappenbeck et al., 1994; Godsel et al., 2005), the PKA inhibitor H89 fails to disrupt DP border localization in keratinocytes (Bass-Zubek et al., 2008; Supplemental Figure 3C). In addition, phosphorylation of CREB, a major downstream target of PKA, is not significantly changed upon Pkp3 ablation in SCC9 and A431 cells (Supplemental Figure S3, A and B). These observations suggest that the cAMP/PKA pathway does not contribute directly to desmosome assembly. Because the only other major cAMP target is EPAC, we tested whether its activator, 8-CPT-2Me-cAMP restores, DP at the sites of cell–cell contacts in Pkp3-deficient cells. As shown in Figure 6, A and B, EPAC stimulation rescued DP accumulation at cell–cell borders in Pkp3- silenced cells. Therefore we proceeded to determine whether the EPAC downstream effector Rap1 GTPase is involved in Pkp3-mediated desmosome assembly. To discern whether Pkp3 KD affected Rap1 activity, we performed pull-down assays for GTP-bound Rap1 in normal human keratinocytes after a calcium switch. Consistent with previous data showing that Rap1 activity is elevated in keratinocytes switched to high calcium (D'silva et al., 2003), in control cells, Rap1 activity increased 5 and 60 min after the calcium switch, whereas no such increase was observed in Pkp3 KD cells (Figure 6C). Finally, overexpression of wtRap1-GFP (Figure 6D) efficiently rescued DP border localization in Pkp3 KD cells (Figure 6, E and F).


Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.

Todorovic V, Koetsier JL, Godsel LM, Green KJ - Mol. Biol. Cell (2014)

Pkp3-dependent Rap1 activation is required for normal DP border localization. (A) Immunofluorescence staining showing the effects of EPAC activator (8-CPT-2Me-cAMP) treatment on DP border localization in control and Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Average DP border intensity for experiments in A. Bars represent mean ± SEM. ns, p > 0.05; ***p < 0.001 (ANOVA, Bonferroni). (C) Western blot showing the levels of GTP-bound (active) Rap1 in NHEK cells treated as indicated. Active Rap1 was pulled down using GST-RalGDS. Rap1 pull-down bands were normalized to total Rap1 using ImageJ. Ratio between activated Rap1 in control and Pkp3 KD cells shows a lack of increase in active Rap1 in Pkp3 KD cells upon calcium switch at 5 and 60 min. (D) Western blot showing the ectopic expression of GFP-tagged Rap1a in SCC9 cells compared with GFP-only control plasmid. nt, no transfection. (E) Immunofluorescence staining of DP showing the recovery of DP at the borders of Rap1-GFP– transfected Pkp3 KD cells. Note the lack of recovery in GFP-only cells. GFP and Rap1-GFP transfection efficiency is shown in the insets marked with white rectangles. Stars represent the cells that are GFP positive in the corresponding inset. Scale bar, 20 μm. (F) Average pixel intensities of DP at the borders of the cells in E. Bars represent mean ± SEM. ns, p >0.05, ***p < 0.001 (ANOVA, Bonferroni).
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Figure 6: Pkp3-dependent Rap1 activation is required for normal DP border localization. (A) Immunofluorescence staining showing the effects of EPAC activator (8-CPT-2Me-cAMP) treatment on DP border localization in control and Pkp3 KD SCC9 cells. Scale bar, 20 μm. (B) Average DP border intensity for experiments in A. Bars represent mean ± SEM. ns, p > 0.05; ***p < 0.001 (ANOVA, Bonferroni). (C) Western blot showing the levels of GTP-bound (active) Rap1 in NHEK cells treated as indicated. Active Rap1 was pulled down using GST-RalGDS. Rap1 pull-down bands were normalized to total Rap1 using ImageJ. Ratio between activated Rap1 in control and Pkp3 KD cells shows a lack of increase in active Rap1 in Pkp3 KD cells upon calcium switch at 5 and 60 min. (D) Western blot showing the ectopic expression of GFP-tagged Rap1a in SCC9 cells compared with GFP-only control plasmid. nt, no transfection. (E) Immunofluorescence staining of DP showing the recovery of DP at the borders of Rap1-GFP– transfected Pkp3 KD cells. Note the lack of recovery in GFP-only cells. GFP and Rap1-GFP transfection efficiency is shown in the insets marked with white rectangles. Stars represent the cells that are GFP positive in the corresponding inset. Scale bar, 20 μm. (F) Average pixel intensities of DP at the borders of the cells in E. Bars represent mean ± SEM. ns, p >0.05, ***p < 0.001 (ANOVA, Bonferroni).
Mentions: The secondary messenger cAMP is responsible for regulating two major signaling axes: protein kinase A (PKA) activation and activation of the Rap1 pathway through the guanine exchange factor (GEF) exchange protein directly activated by cAMP (EPAC; Kopperud et al., 2003; Cheng et al., 2008). Despite our early findings that FSK treatment leads to DP phosphorylation and enhances its accumulation at cell–cell interfaces (Stappenbeck et al., 1994; Godsel et al., 2005), the PKA inhibitor H89 fails to disrupt DP border localization in keratinocytes (Bass-Zubek et al., 2008; Supplemental Figure 3C). In addition, phosphorylation of CREB, a major downstream target of PKA, is not significantly changed upon Pkp3 ablation in SCC9 and A431 cells (Supplemental Figure S3, A and B). These observations suggest that the cAMP/PKA pathway does not contribute directly to desmosome assembly. Because the only other major cAMP target is EPAC, we tested whether its activator, 8-CPT-2Me-cAMP restores, DP at the sites of cell–cell contacts in Pkp3-deficient cells. As shown in Figure 6, A and B, EPAC stimulation rescued DP accumulation at cell–cell borders in Pkp3- silenced cells. Therefore we proceeded to determine whether the EPAC downstream effector Rap1 GTPase is involved in Pkp3-mediated desmosome assembly. To discern whether Pkp3 KD affected Rap1 activity, we performed pull-down assays for GTP-bound Rap1 in normal human keratinocytes after a calcium switch. Consistent with previous data showing that Rap1 activity is elevated in keratinocytes switched to high calcium (D'silva et al., 2003), in control cells, Rap1 activity increased 5 and 60 min after the calcium switch, whereas no such increase was observed in Pkp3 KD cells (Figure 6C). Finally, overexpression of wtRap1-GFP (Figure 6D) efficiently rescued DP border localization in Pkp3 KD cells (Figure 6, E and F).

Bottom Line: Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly.We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing.These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.

Show MeSH
Related in: MedlinePlus