Analysis of T Cell Receptor-Induced Signaling Modules by Mass Spectrometry: NFAT Interactions and the CBM-Complex. InauguralDissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by CHRISTIAN GABRIEL from Leipzig, Germany 2016 EIDESSTATTLICHE ERKLÄRUNG Diese Arbeit wurde vom 15.10.2010 bis zum 15.01.2016 am Deutschen Rheuma- Forschungszentrum in der Arbeitsgruppe von Prof. Dr. Ria Baumgrass angefertigt. 1. Gutachter: Prof. Dr. Ria Baumgrass 2. Gutachter: Prof. Dr. Rupert Mutzel Disputation am: 14. April 2016 i EIDESSTATTLICHE ERKLÄRUNG Eidesstattliche Erklärung Hiermit erkläre ich, dass ich die vorliegende Dissertation selbstständig angefertigt habe. Ausgenommen hiervon sind - wie in Kapitel 2 kenntlich gemacht - die massenspektrometrischen und bioinformatischen Analysen, die in enger Kooperation mit den genannten Wissenschaftlern durchgeführt wurden. Diese Arbeit wurde ohne unzulässige Hilfsmittel angefertigt. Ich habe keine als die hier angegebenen Hilfsmittel und Quellen verwendet. Diese Arbeit wurde in gleicher oder ähnlicher Form noch keiner anderen Prüfungsbehörde vorgelegt. ii TABLE OF CONTENTS Table of Contents Eidesstattliche Erklärung ..........................................................................................................................ii Table of Contents .................................................................................................................................... iii 1. Introduction ..................................................................................................................................... 1 1.1. The Role of T Cells in the Immune System .............................................................................. 1 1.2. The Activation of T Cells. ......................................................................................................... 2 T Cell Stimulation in vitro ................................................................................................ 3 1.3. T Cell Receptor Signaling ......................................................................................................... 3 From T Cell Receptor Stimulation to Phospholipase-C Activation .................................. 3 PKCθ Activation Requires Signaling through the Co-Receptor ........................................ 5 Activation of AP1 Family Proteins through the Action of MAP Kinases .......................... 5 The CBM-Complex Governs the Activation of the Canonical NFκB Pathway in T Cells... 6 1.3.4.1. The CBM-Complex and IKK Activation ..................................................................... 6 1.3.4.2. Post-Translational Modifications Regulate the Activity of the CBM-complex ........ 9 1.3.4.3. CBM-Complex Defects Lead to Immunodeficiency or Cancer Development ........ 10 Calcium Signaling Controls the Activation and Function of NFAT ................................. 11 1.3.5.1. Calcium Influx Triggers the Activation of Calcineurin ........................................... 11 1.3.5.2. NFAT Proteins in Health and Disease .................................................................... 12 1.3.5.3. The Family of NFAT Transcription Factors. ............................................................ 12 1.3.5.4. Structure and Regulation of NFAT Activity ............................................................ 15 1.3.5.5. NFAT Function and Its Interaction with the Transcription Factor AP1.................. 16 1.3.5.6. In the Absence of AP1 Activation, NFAT Promotes T Cell Anergy ......................... 17 1.3.5.7. Interaction of NFAT with Further Transcription Factors ....................................... 18 1.4. Mass Spectrometry to Investigate Protein Complexes ......................................................... 20 MS in Proteomics: Isotope Labeling .............................................................................. 20 Analysis of Protein Complexes by CoIP-MS ................................................................... 22 1.5. The Jurkat Cell Line ................................................................................................................ 24 1.6. Goals of this Thesis ................................................................................................................ 25 2. Material and Methods ................................................................................................................... 26 2.1. Material ................................................................................................................................. 26 Cells ............................................................................................................................... 26 SILAC Media ................................................................................................................... 26 Vectors and Constructs ................................................................................................. 26 Chemicals, Including Peptides and Proteins .................................................................. 28 Pre-made Buffers, Solutions and Stocks ....................................................................... 29 iii TABLE OF CONTENTS Home-made Buffers and Media .................................................................................... 30 Enzymes ......................................................................................................................... 31 Kits ................................................................................................................................. 32 Antibodies...................................................................................................................... 32 Disposables .................................................................................................................... 33 Hardware ....................................................................................................................... 33 Software ........................................................................................................................ 34 2.2. Methods ................................................................................................................................ 34 Methods in Cell Biology ................................................................................................. 34 2.2.1.1. Cell Stimulation ..................................................................................................... 34 2.2.1.2. SILAC Labeling of Jurkat Cells ................................................................................ 34 2.2.1.3. Nucleofection ........................................................................................................ 34 2.2.1.4. Virus Production .................................................................................................... 35 2.2.1.5. Establishment of Stably Transduced Cell Lines by Retroviral Transduction .......... 35 2.2.1.6. Establishment of Stable Transduced Cell Lines by Nucleofection/Selection ........ 35 2.2.1.7. Establishment of Knock-out Cell Lines by CRISPR/CAS9 ........................................ 35 2.2.1.8. Proximity Ligation Assay ........................................................................................ 36 Methods in Protein Biochemistry .................................................................................. 38 2.2.2.1. SDS-PAGE/ Western Blot ....................................................................................... 38 2.2.2.2. Co-Immunopurification ......................................................................................... 39 BCL10 Experiments ................................................................................................................ 39 NFAT Experiments ................................................................................................................. 40 2.2.2.3. MS Measurement .................................................................................................. 41 Bioinformatic Methods.................................................................................................. 41 2.2.3.1. Identification of Peak Regions from Public ChIP-Seq Datasets ............................. 41 2.2.3.2. Identification of Transcription Factor Binding Site Enrichment ............................ 41 2.2.3.3. Identification of Pairs of Transcription Factor Binding Sites ................................. 42 3. Results ........................................................................................................................................... 43 3.1. Characterization of the CBM-complex by Mass Spectrometry ............................................. 43 Generation of a Cell Line that Stably Expresses Epitope-Tagged BCL10 ....................... 43 Isolation of the CBM-Complex via Epitope-Tagged BCL10 ............................................ 44 Isotope Labeling of Jurkat Cells using SILAC .................................................................. 45 Mass Spectrometric Analysis of BCL10 Containing Protein Complexes ........................ 46 TRAF2 and HOIP Interact with BCL10 after TCR Stimulation......................................... 49 3.2. Identification of NFAT Interaction Partners .......................................................................... 52 Establishment of Cell Lines