Regulation of PKP1´s function in intercellular adhesion, proliferation and barrier formation Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Naturwissenschaftlichen Fakultät I – Biowissenschaften – der Martin-Luther-Universität Halle-Wittenberg, vorgelegt von Frau Katrin Rietscher geb. am 18.06.1985 in Räckelwitz Gutachter: Prof. Dr. M. Hatzfeld Prof. Dr. S. Hüttelmaier Prof. Dr. C. Niessen Verteidigungsdatum: 19.04.2018 Meinen Eltern. „Die Endlosigkeit des wissenschaftlichen Ringens sorgt unablässig dafür, daß dem forschenden Menschengeist seine beiden edelsten Antriebe erhalten bleiben und immer wieder von neuem angefacht werden: Die Begeisterung und die Ehrfurcht.“ - Max Planck - CONTENTS 1 ABSTRACT......................................................................................................................................... 1 2 INTRODUCTION .............................................................................................................................. 3 2.1. The epidermis: a network of cytoskeletal elements and cellular junctions .................. 3 The function and structure of the epidermis .................................................................................................... 3 Epithelial cell-cell junctions ...................................................................................................................................... 3 Epidermal differentiation .......................................................................................................................................... 6 2.2. The multifunctional protein PKP1................................................................................................ 9 The structure and expression of PKP1................................................................................................................. 9 The PKP1-associated disease Ectodermal dysplasia-skin fragility syndrome (EDSFS) ............. 11 Function of PKP1 in regulating desmosomal adhesion ............................................................................. 12 Function of PKP1 in the cytoplasm ..................................................................................................................... 13 Function of PKP1 in the nucleus .......................................................................................................................... 15 Post-translational regulation of PKP1 ............................................................................................................... 17 2.3. Epidermal homeostasis: regulation by growth factors ....................................................... 18 Growth factor signaling pathways ...................................................................................................................... 18 Regulation of protein synthesis by growth factors ..................................................................................... 21 2.4. The 14-3-3 proteins ......................................................................................................................... 22 The 14-3-3 protein family: an overview .......................................................................................................... 22 14-3-3 proteins as regulators of PKP functions ............................................................................................ 24 2.5. The aim of the study ......................................................................................................................... 26 3 RESULTS ......................................................................................................................................... 27 3.1. Generation and characterization of PKP1 knockout mice ................................................. 27 Generation and validation of PKP1-KO mice .................................................................................................. 27 PKP1-KO mice reveal growth retardation and die postnatally .............................................................. 30 Desmosome formation and mechanical integrity are disturbed in PKP1-KO epidermis ........... 30 PKP1-KO mice suffer from a defect in the epidermal barrier ................................................................. 34 Desmosomal proteins are compensatorily upregulated in PKP1-KO skin ....................................... 37 Keratin expression is increased in the PKP1-KO skin ................................................................................ 39 Differentiation is mildly altered in PKP1-KO skin ....................................................................................... 40 The localization of AJs is not affected in PKP1-null skin ........................................................................... 42 PKP1-KO keratinocytes recapitulate the effects observed in the epidermis ................................... 43 3.2. Insulin signaling via Akt2 influences PKP1´s subcellular localization and function 49 Generation and characterization of PKP1 phospho-site mutants......................................................... 49 PKP1 localization is regulated by insulin signaling via Akt2 .................................................................. 53 PKP1 phospho-mimetic mutant reduces intercellular adhesion and promotes wound healing ............................................................................................................................................................................................. 55 PKP1 phosphorylation promotes proliferation and confers the capacity for anchorage- independent growth .................................................................................................................................................. 57 3.3. 14-3-3 proteins regulate desmosomal adhesion via PKPs ................................................ 61 14-3-3γ and 14-3-3σ affect intercellular adhesion of keratinocytes ................................................... 61 14-3-3 proteins affect desmosomal protein localization .......................................................................... 64 14-3-3 proteins interact with PKPs in a phosphorylation-dependent manner .............................. 68 14-3-3γ and 14-3-3σ differentially regulate adhesion via PKP1 and PKP3 ..................................... 70 Insulin/Akt2-dependent phosphorylation is required for the PKP1–14-3-3γ association ....... 72 14-3-3γ interferes with desmosomal adhesion by sequestering PKP1 in the cytoplasm .......... 73 14-3-3 proteins differentially modulate PKP dynamics at the desmosome ..................................... 75 4 DISCUSSION ................................................................................................................................... 78 4.1. The role of PKP1 in skin homeostasis ....................................................................................... 78 4.2. Post-translational regulation of PKP1 by insulin signaling .............................................. 82 4.3. Isoform-specific PKP-14-3-3-interactions regulate desmosomal adhesion in keratinocytes ...................................................................................................................................... 88 4.4. PKP1 and β-catenin: comparison of two multifunctional proteins ................................ 93 5 MATERIAL & METHODS ............................................................................................................ 98 5.1. Chemicals and general lab material ........................................................................................... 98 5.2. Phenotypic analysis of PKP1 knockout mice .......................................................................... 98 5.2.1. Animals ............................................................................................................................................................... 98 5.2.2. Documentation of weight development ............................................................................................... 99 5.2.3. Transepidermal water loss (TEWL) assay .......................................................................................... 99 5.2.4. Detection of TJ barrier function in vitro by biotin diffusion assay ........................................... 99 5.2.5. Toluidine blue dye penetration assay ................................................................................................... 99 5.2.6. Preparation and analysis of corneocytes .......................................................................................... 100 5.2.7. Ultrastructural analysis of skin samples ........................................................................................... 100 5.2.8. Preparation of paraffin sections ........................................................................................................... 101 5.2.9. Preparation of cryosections ................................................................................................................... 101 5.2.10. Hematoxylin & Eosin (H&E) staining .............................................................................................. 101 5.2.11. Connective tissue stain .......................................................................................................................... 102 5.2.12. Immunohistochemistry (IHC) of paraffin sections ...................................................................