Redox-mediated regulation of the tyrosine kinase Zap70 DISSERTATION zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) genehmigt durch die Fakultät für Naturwissenschaften der Otto-von-Guericke-Universität von M.Sc. Christoph Thurm geb. am 27.06.1988 in Borna Gutachter: apl. Prof. Dr. Luca Simeoni PD Dr. rer. nat. Marcus Lettau eingereicht am: 02.02.2018 verteidigt am: 06.06.2018 Eigenständigkeitserklärung I. Eigenständigkeitserklärung Christoph Thurm Halberstädter Straße 29 39112 Magdeburg Hiermit erkläre ich, dass ich die von mir eingereichte Dissertation zu dem Thema Redox-mediated regulation of the tyrosine kinase Zap70 selbständig verfasst, nicht schon als Dissertation verwendet habe und die benutzten Hilfsmittel und Quellen vollständig angegeben wurden. Weiterhin erkläre ich, dass ich weder diese noch eine andere Arbeit zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) an anderen Einrichtungen eingereicht habe. Magdeburg, den 02.02.2018 ____________________________________ M.Sc. Christoph Thurm II ACKNOWLEDGEMENTS II. Acknowledgements Firstly, I would like to express my sincere gratitude to my supervisor Prof. Dr. Luca Simeoni. His extraordinary support during my PhD thesis together with his motivation and knowledge enabled me to pursue my dream. I could not have imagined having a better mentor. Furthermore, I would like to thank Prof. Dr. Burkhart Schraven for giving me the opportunity to work in his institute. His support, ideas, and the lively discussions promoted me to develop as a scientist. Special thanks go also to the whole AG Simeoni/Schraven - Ines, Camilla, Matthias, and Andreas - for the help with experiments, the discussions, and the fun we had. This helped to sustain also the longest days. Also, I would like to thank the whole IMKI team for the outstanding atmosphere and spirit. I really enjoyed being a part of you. I would like to thank my family and friends who covered my back also during the most painful times. Especially my father who guided me to become the person I am today. Finally, I would like to thank Anne who supported me every single day and blessed me with her patience and her positive attitude. Words cannot express what it means to me to know you by my side. III TABLE OF CONTENT III. Table of content I. EIGENSTÄNDIGKEITSERKLÄRUNG ........................................................................................... II II. ACKNOWLEDGEMENTS .............................................................................................................. III III. TABLE OF CONTENT ................................................................................................................... IV IV. LIST OF FIGURES AND TABLES ................................................................................................ VII V. ABBREVIATIONS .......................................................................................................................... IX VI. ABSTRACT ................................................................................................................................... XII VII. ZUSAMMENFASSUNG ............................................................................................................... XIII 1. INTRODUCTION ............................................................................................................................. 1 1.1 SIGNALING IN EUKARYOTIC CELLS .................................................................................................. 1 1.2 PROTEIN TYROSINE KINASES ......................................................................................................... 2 1.2.1 Receptor Tyrosine Kinases ............................................................................................. 4 1.2.2 Non-receptor tyrosine kinases ......................................................................................... 6 1.3 THE TYROSINE KINASE ZAP70 ........................................................................................................ 8 1.3.1 Function of Zap70 ............................................................................................................ 9 1.3.1.1 T cells and TCR signaling ......................................................................................................... 9 1.3.1.2 T-cell development .................................................................................................................. 11 1.3.2 Zap70 in diseases .......................................................................................................... 13 1.3.2.1 Immunodeficiency ................................................................................................................... 13 1.3.2.2 Autoimmunity .......................................................................................................................... 15 1.3.2.3 Leukemia ................................................................................................................................ 16 1.3.3 Regulation of Zap70 ...................................................................................................... 17 1.3.3.1 Membrane recruitment ............................................................................................................ 17 1.3.3.2 Tyrosine phosphorylation ........................................................................................................ 19 1.3.3.3 Ubiquitination .......................................................................................................................... 22 1.3.3.4 Oxidation ................................................................................................................................. 23 1.4 REGULATION OF TYROSINE KINASES BY OXIDATION ....................................................................... 24 1.4.1 Cellular sources of ROS and RNS ................................................................................ 26 1.4.2 Modulation of the activity of tyrosine kinases by oxidation ............................................ 29 1.4.2.1 Direct regulation of kinases via ROS....................................................................................... 29 1.4.2.2 Regulation of phosphatases by ROS ...................................................................................... 30 1.4.2.3 Perspectives in therapy ........................................................................................................... 30 1.5 AIMS OF THE STUDY .................................................................................................................... 32 2. MATERIALS AND METHODS ...................................................................................................... 33 2.1 CHEMICALS, ANTIBODIES, AND OTHER REAGENTS ......................................................................... 33 2.1.1 Chemicals ...................................................................................................................... 33 2.1.2 Antibodies for western blot and immunoprecipitation .................................................... 34 2.1.3 Antibodies for intracellular FACS analysis .................................................................... 34 2.1.4 Antibodies for stimulation .............................................................................................. 34 2.2 PLASMIDS ................................................................................................................................... 35 2.3 BUFFERS AND SOLUTIONS ............................................................................................................ 35 2.4 CELL CULTURE ............................................................................................................................ 38 2.4.1 Jurkat cell culture ........................................................................................................... 38 2.4.2 HEK293T cell culture ..................................................................................................... 38 2.4.3 Treatments with inhibitors and antioxidants .................................................................. 38 2.5 SITE DIRECTED MUTAGENESIS ...................................................................................................... 39 2.6 TRANSFECTION ........................................................................................................................... 41 2.6.1 Transfection by calcium phosphate ............................................................................... 41 2.6.2 Transfection by electroporation ..................................................................................... 42 2.7 CELL STIMULATION ...................................................................................................................... 42 2.7.1 Stimulation with soluble Antibodies (sAbs) .................................................................... 42 2.7.2 Ca2+-Flux measurements ............................................................................................... 42 2.7.3 Stimulation with antibodies immobilized on microspheres (iAbs) .................................. 42 IV TABLE OF CONTENT 2.8 WESTERN BLOTTING .................................................................................................................... 43 2.8.1 General procedure ........................................................................................................