A role of plakophilin 3 in keratinocyte proliferation

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Desmosomes are intercellular adhesive contacts that are especially abundant in tissues prone to mechanical strain such as the skin and the heart. Our data suggest that members of the plakophilin family of desmosomal proteins control desmosome adhesive strength, size and number. Whereas plakophilin 1 strengthens intercellular adhesion and increases desmosome size in vivo and in vitro, plakophilin 3 renders desmosomes more dynamic. In agreement, our mouse knockout studies reveal severe skin fragility in plakophilin 1 KO mice with postnatal lethality. Moreover, we have shown that plakophilins 1 and 3 act as switches between desmosomal adhesion, cell proliferation, cell migration and anchorage independent growth. Cytoplasmic plakophilin 1 stimulates proliferation by associating with the translation initiation complex to increase protein biosynthesis. Activation of these regulatory functions depends on plakophilin 1 phosphorylation by the Akt2 kinase downstream of IGF1/insulin signaling. Phosphorylated plakophilin 1 becomes trapped in the cytoplasm by 14-3-3 which interferes with its incorporation into desmosomes. We found that plakophilin 3 knockout keratinocytes reveal significantly reduced proliferation rates, in agreement with growth retardation of plakophilin 3 KO mice, whereas its overexpression promotes proliferation of keratinocytes. Our comparative gene expression analysis revealed that 30% of all genes downregulated in plakophilin 3 KO cells are cell cycle related, many of them being E2F targets. Moreover, we find that plakophilin 3 translocates to the mitotic spindle. The identification of several putative interacting proteins essential for mitotic spindle formation and dynamics suggests an additional role in cell cycle progression. Based on these data we propose the hypothesis that plakophilin 3 is involved in cell cycle control by (a) regulating S phase entry via control of E2F/Rb activity and (b) regulating the mitotic spindle in association with microtubule binding proteins. Our project aims to verify this hypothesis by validation of E2F target gene regulation and an analysis of the molecular mechanism by which plakophilin 3 might influence E2F/Rb activity to promote S-phase entry. Moreover, plakophilin 3 localization and protein interactions during mitosis and mechanisms controlling its changes in localization and protein interactions in a cell cycle dependent manner will be studied.We expect that our studies will have a major impact on understanding the interplay between desmosomal adhesion and proliferation which has to be tightly balanced to guarantee epidermal homeostasis. Moreover, we will gain insight into the role of plakophilin 3 in modulating desmosomes to allow for mobility without interfering with cohesion during mitosis.