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

嗜血小板蛋白4 （PKP4, p0071）是粘附连接蛋白（AJ）的一种，其表达仅限于表皮基底细胞，而相关的p120在所有角质形成细胞中表达，无论其分化程度如何。此外，PKP4在细胞分裂过程中影响rho信号传导。为了更好地理解PKP4的功能，我们建立了一个由野生型（WT）、PKP4敲除（PKP4- ko）和“拯救”细胞系（PKP4在PKP4- ko细胞系中重新表达）组成的模型系统。利用该模型系统，我们对PKP4功能进行了初步研究，结果表明PKP4的缺失导致肌动蛋白细胞骨架组织发生巨大变化，应力纤维增加，皮质肌动蛋白减少。这与张力降低和细胞接触改变有关。该项目的目标是揭示这些变化背后的分子机制。重点将放在PKP4在调控Rho GTPases中的作用，特别是RhoA。首先，确定PKP4对Rho GTPases局部活性的影响。此外，我们将研究PKP4如何通过与上游调节因子的相互作用来调节Rho GTPases的活性。为此，选择了一组鸟嘌呤交换因子（GEF）和GTPase激活蛋白（GAP）进行了详细的表征。最后，下游效应将以肌动球蛋白组织、肌球蛋白活性和组织张力的产生为特征。机械信号反过来通过Hippo/YAP/TAZ活性控制增殖和分化。在这个项目中产生的数据将提高我们对基底角质形成细胞的增殖和分层是如何被控制的理解。这对各种皮肤疾病有影响，其中增殖和分化之间的平衡受到干扰，以及皮肤老化和/或干细胞维持的变化。

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.