Julia Lindqvist Lindqvist Julia To divide or differentiate? divide differentiate? or To Nestin-mediated regulation of Cdk5 in cell fate decisions Cdk5 of in cell fate regulation Nestin-mediated To divide or differentiate? Nestin-mediated regulation of Cdk5 in cell fate decisions Julia Lindqvist ISBN 978-952-12-3238-1 Department of Biosciences Painosalama Oy – Turku, Finland 2015 Faculty of Science and Engineering, Åbo Akademi University Turku Centre for Biotechnology, University of Turku and Åbo Akademi University 2015 Turku Doctoral Programme of Biomedical Sciences Finland, 2015 To divide or differentiate? Nestin-mediated regulation of Cdk5 in cell fate decisions Julia Lindqvist Department of Biosciences Faculty of Science and Engineering, Åbo Akademi University Turku Centre for Biotechnology, University of Turku and Åbo Akademi University Turku Doctoral Programme of Biomedical Sciences Finland, 2015 From the Department of Biosciences, Faculty of Science and Engineering, Åbo Akademi University, Turku Centre for Biotechnology, University of Turku and Åbo Akademi University and Turku Doctoral Programme of Biomedical Sciences Supervised by Professor John Eriksson Department of Biosciences, Faculty of Science and Engineering, Åbo Akademi University Turku Centre for Biotechnology, University of Turku and Åbo Akademi University Turku, Finland Reviewed by Professor Thomas Magin Institute of Biology and Translational Center for Regenerative Medicine University of Leipzig Leipzig, Germany Docent Maria Vartiainen Institute of Biotechnology University of Helsinki Helsinki, Finland Opponent Dr. Karen M. Ridge Feinberg School of Medicine Northwestern University Chicago, USA ISBN 978-952-12-3239-8 Painosalama Oy – Turku, Finland 2015 To my family and friends TABLE OF CONTENTS List of Original Publications ...................................................................................................... 6 Abbreviations ............................................................................................................................. 7 Abstract....................................................................................................................................... 9 Introduction ............................................................................................................................. 10 Review of the Literature ........................................................................................................... 12 1 Cyclin-dependent kinase 5 in development and disease .......................................................... 12 1.1 The molecular structure of Cdk5 ......................................................................................... 12 1.2 Mechanisms of Cdk5 activation ........................................................................................... 13 1.3 Developmental and physiological functions of Cdk5 ....................................................... 17 1.4 Pathological deregulation of Cdk5 ...................................................................................... 24 2 The intermediate filament protein nestin in cell signaling ...................................................... 32 2.1 Intermediate filament essentials: structure and dynamics ............................................... 32 2.2 Post-translational regulation of intermediate filament assembly ................................... 36 2.3 Intermediate filaments as signaling scaffolds..................................................................... 39 3 Myogenesis ...................................................................................................................................... 44 3.1 Skeletal muscle stem cells and muscle differentiation ...................................................... 45 3.2 Muscle repair processes ......................................................................................................... 46 3.3 Laboratory models in muscle research ............................................................................... 47 Aims of the Study ..................................................................................................................... 48 Experimental Procedures ......................................................................................................... 49 1 Cell culture methods and experimental treatments .................................................................. 49 1.1 Cell lines................................................................................................................................... 49 1.2 Experimental treatments ....................................................................................................... 49 1.3 Transient transfections and plasmids ................................................................................. 50 1.4 Cell proliferation assays......................................................................................................... 50 2 Mouse strains and tissue preparation .......................................................................................... 51 2.1 Primary muscle myoblast cultures....................................................................................... 51 2.2 Myofiber isolation .................................................................................................................. 52 2.3 TA-muscle injury ................................................................................................................... 52 3 Immunoprecipitation and cell fractionation .............................................................................. 52 4 Western blotting ............................................................................................................................. 52 5 Immunocytochemistry and immunohistochemistry ................................................................ 53 6 Flow cytometry ................................................................................................................................ 54 7 Cdk5 kinase activity ........................................................................................................................ 54 8 In vitro phosphorylation and mass spectrometry ...................................................................... 54 9 Statistical analysis ............................................................................................................................ 55 Results and Discussion ............................................................................................................. 56 1 Cdk5-nestin crosstalk adjusts myogenic differentiation (I) ..................................................... 56 1.1 Nestin fine-tunes the pace of muscle differentiation ........................................................ 56 1.2 The importance of the nestin scaffold in regulation of p35 turnover in myoblasts ..... 57 1.3 Cdk5 talks back: self-regulation through nestin ................................................................ 59 2 Nestin regulates skeletal muscle homeostasis and regeneration in vivo (II) ......................... 60 2.1 Gender and age-dependent variability in myoblast p35 expression ............................... 60 2.2 Nestin-/- mice have less lean mass and show signs of spontaneous muscle regeneration ......................................................................................................................................................... 62 2.3 Muscle healing after injury is delayed in nestin-/- mice .................................................... 63 3 Cdk5 controls prostate cancer cell proliferation through Akt and AR (III) .......................... 65 3.1 Nestin - a Cdk5 scaffold in prostate cancer? ...................................................................... 65 3.2 Cdk5 phosphorylates AR and guides its target gene specificity ...................................... 66 3.3 Cdk5 regulates prostate cancer cell proliferation .............................................................. 67 3.4 Complex Cdk5-Akt-androgen interplay in prostate cancer cells .................................... 68 Concluding Remarks ................................................................................................................ 73 Acknowledgements................................................................................................................... 74 References ................................................................................................................................. 76 Original Publications ............................................................................................................... 89 List of Original Publications LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following original publications and manuscripts. They are referred to in the text by their roman numerals. The original publications have been reproduced with permission of the copyright holders. In addition, the thesis includes unpublished data. I. Pallari H.-M., Lindqvist J., Torvaldson E., Ferraris S.E., He T., Sahlgren C., Eriksson J.E. Nestin as a regulator of Cdk5 in differentiating myoblasts Mol Biol Cell. 2011 May; 22(9):1539-49 II. Lindqvist J.*, Torvaldson E.*, Gullmets J., Karvonen H., Taimen P., Eriksson J.E. The intermediate filament protein nestin is required for skeletal muscle physiology and regeneration Manuscript III. Lindqvist J., Imanishi S.I.*, Torvaldson E.*, Malinen M.,