Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.
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.
Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.Show MeSH
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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).
Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.