Characterization of Plakophilin 4 as a key regulator of Rho signaling in epidermal keratinocytes
Final Report Abstract
Plakophilin 4 (PKP4, p0071) is a protein of adherens junctions (AJs) whose expression is restricted to the proliferating cells of the epidermis. To unravel the specific functions of PKP4 in keratinocytes, we generated a model system with wild-type (WT), PKP4 knockout (PKP4-KO), and "rescue" keratinocyte cell lines (re-expression of PKP4 in the PKP4-KO line). Using this model system, we show that PKP4-KO cells failed to develop a cortical actin ring. This prevented AJ maturation and caused a loss of tissue tension. Instead, PKP4-depleted cells displayed increased stress fibers. In order to uncover the molecular mechanisms underlying these changes we focused on the role of PKP4 in regulating Rho GTPases, in particular RhoA. We show that RhoA localization at AJs depended on PKP4 and was required for the local activation of a RhoA-ROCK2- MLCK-MLC2 axis and organization of actin into a cortical ring. To investigate how PKP4 modulates the activity of Rho GTPases via interactions with upstream regulators, a subset of guanine-exchange factors (GEF) and GTPase activating proteins (GAP) was selected for a detailed characterization. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization. Mechanosignaling controls proliferation and differentiation via the Hippo signalling pathway. Loss of PKP4 increased proliferation but at the same time induced premature epidermal differentiation. Mechanistically, PKP4 interacted with several components of the Hippo pathway including the YAP/TAZ transcriptional co-activators which are known as important regulators of growth and differentiation. PKP4 controlled nuclear YAP localization and target gene expression as well as its cell-contact association suggesting a role in balancing YAP/TAZ localization and functions. YAP depletion on the other hand reduced PKP4 levels and keratinocyte adhesion indicative of a feedback mechanism between PKP4 and YAP that controls adhesion, proliferation and differentiation by balancing YAP functions. The continuous balance of proliferation and differentiation is essential for skin homeostasis. Our data indicate that PKP4 plays a crucial role in this context by dynamically regulating Hippo signaling through its downstream effector YAP. Linking PKP4 to the Hippo signaling pathway adds an important new aspect of AJs in regulating cell growth and differentiation suggesting a putative role of PKP4 in modulating Hippo signaling and cell adhesion in cancer.
