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: To address whether Pkp3 governs Rap1 activity through a physical association, we first gauged whether the two proteins exist in close proximity in situ, using a proximity ligation assay (PLA). Our results indicate that Pkp2 and Pkp3 are both found in close association with DP, as predicted, whereas only Pkp3 is found to be in close proximity with Rap1 (Figure 7A). To test biochemically whether Pkp3 interacts with Rap1, we performed immunoprecipitation experiments. The data show Pkp3 being specifically precipitated with anti-Rap1 antibody, whereas this is not the case with Pkp2 (Figure 7B). Intriguingly, the data also show that E-cad, a known functional interactor of Rap1 (Retta et al., 2006; Li et al., 2010), loses its ability to complex with Rap1 upon Pkp3 ablation. Further, in contrast to the largely distinct localization of E-cad and DP, E-cad and Pkp3 substantially colocalize at cell–cell borders, with a concentration of colocalizing signals at the cell margins (Figure 7C). E-cad knockdown did not have the same disruptive effect on Pkp3-Rap1 interaction or the interaction between Rap1 and its main activator, EPAC, suggesting that Pkp3 is required for Rap1 interaction with E-cad, but E-cad is not required for Pkp3-Rap1 interaction (Figure 7D). To test whether the activity of the Rap1 GEF EPAC was required for the maintenance of a Rap1/Pkp3/E-cad complex, we inhibited EPAC activity by using its well-established chemical inhibitor ESI-09 (Almahariq et al., 2013). In the absence of EPAC activity, E-cad is excluded from a complex that still contains Pkp3 and Rap1, supporting the idea that Pkp3 provides a scaffold for binding and activation of Rap1 required for E-cad association (Figure 7E).
Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.