Analyse von T-Zell-Rezeptor-induzierten Signalmodulen: NFAT-Interaktionen und der CBM-Komplex

1\. Introduction 1 1.1. The Role of T Cells in the Immune System 1 1.2. The Activation of T Cells. 2 1.2.1. T Cell Stimulation in vitro 3 1.3. T Cell Receptor Signaling 3 1.3.1. From T Cell Receptor Stimulation to Phospholipase-C Activation 3 1.3.2. PKCθ Activation Requires Signaling through the Co-Receptor 5 1.3.3. Activation of AP1 Family Proteins through the Action of MAP Kinases 5 1.3.4. 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 1.3.5. 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 1.4.1. MS in Proteomics: Isotope Labeling 20 1.4.2. 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 2.1.1. Cells 26 2.1.2. SILAC Media 26 2.1.3. Vectors and Constructs 26 2.1.4. Chemicals, Including Peptides and Proteins 28 2.1.5. Pre-made Buffers, Solutions and Stocks 29 2.1.6. Home-made Buffers and Media 30 2.1.7. Enzymes 31 2.1.8. Kits 32 2.1.9. Antibodies 32 2.1.10. Disposables 33 2.1.11. Hardware 33 2.1.12. Software 34 2.2. Methods 34 2.2.1. 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 2.2.2. 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 2.2.3. 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 3.1.1. Generation of a Cell Line that Stably Expresses Epitope-Tagged BCL10 43 3.1.2. Isolation of the CBM-Complex via Epitope-Tagged BCL10 44 3.1.3. Isotope Labeling of Jurkat Cells using SILAC 45 3.1.4. Mass Spectrometric Analysis of BCL10 Containing Protein Complexes 46 3.1.5. TRAF2 and HOIP Interact with BCL10 after TCR Stimulation 49 3.2. Identification of NFAT Interaction Partners 52 3.2.1. Establishment of Cell Lines that Stably Express Biotin- Tagged NFAT Proteins 52 3.2.1.1. Choice of Epitope-Tag and Vector System 52 3.2.1.2. Stable Transfection of Jurkat Cells with Epitope-Tagged NFAT Isoforms 54 3.2.2. Characterization of NFAT Overexpressing Cell Lines 56 3.2.2.1. Cellular Localization of Epitope-Tagged NFAT Proteins 56 3.2.2.2. Phosphorylation of Epitope-Tagged NFAT Proteins 57 3.2.3. Purification of NFAT Proteins by the Help of the AVITEV Tag 60 3.2.4. Co-Purification of Known NFAT Interacting Proteins 61 3.2.5. Mass Spectrometric Analyses of NFAT Containing Protein Complexes 63 3.2.5.1. Design of CoIP-MS Experiments to Identify Proteins that Interact with NFAT 63 3.2.5.2. Overview of MS Analyses of NFAT Containing Protein Complexes 64 3.2.5.3. Protein-Protein Interactions Involving NFATc1S and NFATc1L 68 3.2.5.4. Protein-Protein Interactions Involving NFATc2 70 3.2.6. NFATc2 and Ikaros Interact in Primary Human T Helper Cells after TCR Stimulation 72 3.2.7. NFATc2 Interactions Divide into DNA-Dependent and DNA-Independent 76 3.2.8. Co-Occurrence of NFAT DNA Binding Motifs with those of other Transcription Factors 78 3.2.8.1. Binding Motifs for AP-1 proteins, RUNX and Ets-Factors Are Enriched in Regions of NFAT Binding 78 3.2.8.2. Pairs of Binding Motifs for NFAT and AP1, CRE and RUNX Are Found Preferentially in a Distinct Distance and Orientation 80 4\. Discussion 84 4.1. HOIP: A Novel Regulator of the CBM-Complex 84 4.1.1. Isolation of the CBM-Complex: Known and Suspected Components 84 4.1.2. The LUBAC Constituents HOIP and HOIL1Associate with the CBM Complex 85 4.1.3. Expression of HOIP Is Necessary for TCR Induced NFκB Activation and IL-2 Expression 87 4.1.4. The LUBAC and the CBM: Open Questions 88 4.2. Identification of NFAT Interaction Partners 91 4.2.1. General Remarks 91 4.2.1.1. Influence of Overexpression and the Biotin-Tag on NFAT Localization and Phosphorylation 91 4.2.1.2. JUN Is Likely to Be Cleaved by TEV Protease 92 4.2.1.3. Discrepancy between the Amount of NFATc1S and NFATc1L Interactors 92 4.2.1.4. Defining a Cut-Off for Potential and Confident Interactors 93 4.2.1.5. Absence of Proof Is not a Proof of Absence 94 4.2.1.6. Towards the Confidence and Nature of Suggested Interactions 94 4.2.1.7. Bioinformatics Suggest Cooperative DNA Binding of NFAT with RUNX- and CRE-binding Proteins 97 4.2.2. Implications of Selected Interactions 98 4.2.2.1. CREB1 and NFAT-CRE Binding Motifs 98 4.2.2.2. RUNX and NFAT: Cooperative DNA Binding? 100 4.2.2.3. Ikaros: Repressor of NFAT Transactivation? 100 4.2.2.4. IRF2BP1 and IRF2BP2: Two Further Suppressors of NFATc2 Transactivation? 101 4.2.2.5. SATB1: Chromatin Opening at Sites of NFAT Binding? 102 4.2.2.6. SCAI, WDR48 and RPTOR: DNA Independent NFAT-Interactors 102 4.2.2.7. CHEK1, NEK6, NEK7 and PLK: Potential NFAT Kinases? 103 4.2.2.8. 14-3-3 Proteins, RANBP9 and CABIN-1: Regulators of NFAT Phosphorylation and Activation 103 4.2.3. Concluding Remarks 104 4.2.4. Outlook 104A T cell is activated by the binding of a specific antigen to its T cell receptor. Subsequently, signaling modules integrate information from the antigen receptor, environment and cellular background to produce an adequate outcome. Mechanistically, the activation of diverse transcription factors drives and shapes the cellular reaction in most cases. The transcription factors nuclear factor of activated T cells (NFAT) and nuclear factor of κ light chain enhancer of activated B cells (NFκB) play a pivotal role in T cell biology. Aberrations in their activation pathways can lead to immunodeficiency, autoimmune diseases and cancer development. A complex that consists of CARMA1, BCL10 and MALT1 (CBM-complex) controls the activation of NFκB following TCR ligation by integrating input from different signaling pathways (PKCθ, calcineurin). Employing a combination of co-immunopurification with mass spectrometry, we identified HOIP and HOIL1, two compounds of the linear ubiquitin assembly complex LUBAC, as so far unknown interactors of BCL10. We proved that HOIP interacts with BCL10 after TCR stimulation in Jurkat cells and after PMA/ionomycin stimulation in primary T helper cells. The presence of HOIP, but not its enzymatic activity, is necessary for complete NFκB activation after TCR/co-receptor engagement; a finding that was revealed in recent reports from other groups. Thus, HOIP constitutes a new potential target to modulate T cell activation. However, it remains unclear how LUBAC components are recruited to the CBM signaling complex and how they contribute to the activation of IKK and NFκB. The activation of NFAT transcription factors is a hallmark of T cell activation and a pre-requisite for most T cell effector functions. NFAT readily interacts with other transcription factors. These interactions strongly influence the locus and the outcome of NFAT binding. Via binding to the IL-2 promoter, NFAT in a complex with AP1 promotes IL 2 expression, while a complex of NFAT and FOXP3 represses IL-2 expression. In the absence of interaction partners, NFAT binding to this locus is not detectable. By combining co-immunopurification with mass spectrometry, we identified more than 100 potential previously unknown interaction partners of NFATc1 and NFATc2, including more than 40 transcription factors. We could confirm a range of these interactions in follow-up experiments, including those with Ikaros, CREB1 and RUNX1. Additionally, we identified potential common binding motifs of NFAT with these transcription factors by the use of bioinformatics. Thereby, we found that dimeric NFAT-RUNX and NFAT-CRE binding motifs are highly enriched within genomic regions of NFAT binding in activated cytotoxic T cells. Further experiments, including ChIP-Seq and molecular interaction studies, will reveal how NFAT concerts T cell functions within different T cell subsets and how other proteins influence NFAT’s activity to shape the outcome of T cell activation. This may advance the development of more specific immune- modulatory treatments.T-Zellen werden durch die Bindung eines spezifischen Antigens über ihren T -Zell-Rezeptor (TZR) aktiviert. Die vom Antigenrezeptor kommenden Signale werden in Signalkomplexen verarbeitet, mit zusätzlichen Informationen zu Umgebung und Zellstatus versehen und integriert, um eine adäquate Reaktion der Zelle auszulösen. Diese Reaktion wird in vielen Fällen durch die Aktivierung verschiedener Transkriptionsfaktoren ausgelöst und moduliert. Die Transkriptionsfaktoren Kernfaktor in aktivierten T-Zellen (NFAT) und Kernfaktor des κ-Leichtkettenverstärkers in aktivierten B-Zellen (NFκB) spielen eine entscheidende Rolle bei der T-Zellaktivierung. Anomalien in deren Regulierung führen zu Immundefekten, Autoimmunerkrankungen und Krebs. Die NFκB-Aktivierung nach TZR-Stimulation wird vom CBM-Komplex (für CARMA1-BCL10-MALT1-Komplex) durch die Integration der Einträge verschiedener Signalwege kontrolliert. Durch Kombination von Co-Immunpräzipitation und Massenspektrometrie konnten wir die Proteine HOIP und HOIL1, die Teil des Ubiquitin-Ligase-Komplexes LUBAC sind, als Interaktionspartner von BCL10 identifizieren. Wir konnten zeigen, dass HOIP nach Stimulation sowohl in Jurkat-Zellen als auch in primären humanen T-Helferzellen mit BCL10 interagiert. Das Vorhandensein von HOIP – nicht aber dessen katalytische Aktivität – ist für eine komplette NFκB-Aktivierung nach T-Zellaktivierung essentiell, wie kürzlich erschienene Arbeiten anderer Gruppen offenbart haben. Somit ist HOIP ein neues potentielles Target zur Modulierung der T-Zellaktivierung. Unklar ist jedoch weiterhin, wie der LUBAC-Komplex zum CBM- Komplex rekrutiert wird und wie genau er zur Aktivierung von IKK und NFκB beiträgt. Die Aktivierung von NFAT-Transkriptionsfaktoren ist Voraussetzung für die meisten T-Zell-Effektorfunktionen. NFAT interagiert mit anderen Transkriptionsfaktoren. Diese Interaktionen haben großen Einfluss darauf, an welchen DNA-Sequenzen NFAT bindet und welche Wirkung diese Bindung hervorruft. So hat NFAT beispielsweise im IL-2-Promoterbereich zusammen mit AP1 einen aktivierenden und mit FOXP3 einen inhibierenden Effekt auf die IL-2-Produktion. In Abwesenheit von Interaktionspartnern ist eine Bindung von NFAT im IL-2-Promoterbereich hingegen nicht nachweisbar. Durch Kombination von Co-Immunpräzipitation und Massenspektrometrie konnten wir über 100 bislang unbekannte, potentielle Interaktionspartner von NFATc1 und NFATc2 identifizieren, darunter mehr als 40 Transkriptionsfaktoren. Einige dieser Interaktionen (darunter die mit CREB1, RUNX1 und Ikaros) konnten im Laufe dieser Arbeit verifiziert werden. Zusätzlich haben wir mithilfe bioinformatischer Methoden potentielle gemeinsame Bindungsstellen dieser Transkriptionsfaktoren mit NFAT identifiziert. Hierbei zeigte sich, dass in genomischen Regionen, die von NFAT in aktivierten zytotoxischen T Zellen gebunden werden, dimere NFAT-RUNX und NFAT-CRE Bindemotive stark gehäuft auftreten. Weiterführende Experimente wie ChIP-Seq und molekulare Interaktionsstudien werden das Verständnis dafür schärfen, wie NFAT die T -Zell-Funktionen in verschiedenen T-Zelltypen moduliert, und wie andere Proteine die NFAT-Aktivität beeinflussen um die T-Zell-Aktivierung zu regulieren. Dies könnte die Entwicklung spezifischerer immun-modulierender Therapien vorantreiben