Characterization of Plakophilin 4 as a key regulator of Rho signaling in epidermal keratinocytes

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Plakophilin 4 (PKP4, p0071) is a protein of adherens junctions (AJ) whose expression is restricted to the basal cells of the epidermis, whereas the related p120 is expressed in all keratinocytes regardless of their degree of differentiation. In addition, PKP4 is known to affect Rho-signaling in cytokinesis. To better understand the functions of PKP4, we generated a model system consisting of a wild-type (WT), a PKP4 knockout (PKP4-KO), and a "rescue" cell line (re-expression of PKP4 in the PKP4-KO line). Using this model system, we have performed preliminary studies of PKP4 function showing that loss of PKP4 leads to massive changes in actin cytoskeleton organization with increased stress fibers but decreased cortical actin. This correlates with decreased tension and changes in cell contacts. The goal of the proposed project is to uncover the molecular mechanisms underlying these changes. The focus will be on the role of PKP4 in the regulation of Rho GTPases, in particular RhoA. First, the influence of PKP4 on the local activity of Rho GTPases will be determined. In addition, we will investigate how PKP4 modulates the activity of Rho GTPases via interactions with upstream regulators. For this purpose, a subset of guanine-exchange factors (GEF) and GTPase activating proteins (GAP) were selected for a detailed characterization. Finally, downstream effects will be characterized in terms of actomyosin organization, myosin activity and generation of tissue tension. Mechanosignaling in turn controls proliferation and differentiation via Hippo/YAP/TAZ activity. The data generated in this project will improve our understanding how proliferation and delamination of basal keratinocytes is controlled. This has implications for various skin diseases in which the balance between proliferation and differentiation is disturbed, as well as for changes in skin aging and/or stem cell maintenance.