„Role of STAT3 N-terminal domain and GAS-site recognition in signaling and crosstalk with STAT1 and NF-κB” Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von Master of Science Antons Martincuks aus Riga, Lettland Berichter: Professor Dr. Gerhard Müller-Newen Universitätsprofessor Dr. Martin Zenke Universitätsprofessor Dr. Bernhard Lüscher Tag der mündlichen Prüfung: 23.01.2017 Diese Dissertation ist auf den Internetseiten der Universitätsbibliothek online verfügbar. To my father (28.09.1961 – 26.01.2004) Für meinen Vater (28.09.1961 – 26.01.2004) Моему отцу (28.09.1961 – 26.01.2004) Table of contents Publications and coauthorships 2 Zusammenfassung 4 Abstract 5 I. Introduction 6 1. Cellular signaling 6 2. JAK/STAT pathway 7 2.1 History and pathway overview 7 2.2 Structure and functions of STAT proteins 9 2.3 IL-6/STAT3 signaling 11 2.3.1 IL-6 type cytokines 11 2.3.2 IL-6/STAT3 signal transduction 12 2.3.3 STAT3 in physiology and disease 14 2.3.4 The N-terminal domain of STAT3 in signaling 15 2.4 Crosstalk between STAT1 and STAT3 17 2.4.1 Opposing actions of STAT1 and STAT3 17 2.4.2 Crossregulation of IFNγ/STAT1 and IL6/STAT3 signaling 18 3. STAT3 and NF-κB pathway crosstalk 20 3.1 NF-κB signaling overview 20 3.2 Canonical TNFα signaling 21 3.3 NF-κB role in physiology and cancer 21 3.4 Structure and posttranslational modifications of p65 22 3.5 NF-κB and STAT3 crosstalk 24 4. Nucleocytoplasmic trafficking of proteins 26 4.1 Molecular mechanism of nuclear import and export 26 4.2 Properties of importin-α adaptors 28 4.3 Nuclear trafficking of STAT1 and STAT3 29 5. Aims of the study 30 II. Materials and Methods 32 1. Materials 32 1.1 Chemicals and reagents 32 1.2 Plasmids 32 1.3 qPCR Primers 34 1.4 Mutagenesis primers 34 1.5 Genotyping primers 35 1.6 Antibodies 36 1.6.1 Primary antibodies 36 1.6.2 Secondary antibodies 36 1.7 Eukaryotic cell lines 37 1.7.1 Stably transfected cell lines 38 1.7.2 Growth medium 39 1.7.3 Cytokines and cytokine receptors 39 1.7.4 Transfection reagents 40 1.7.5 Other reagents 40 1.8 Prokaryotic cell lines 40 1.8.1 Cultivation 40 1.9 Antibiotics 41 2. Methods 42 2.1 Molecular biology methods 42 2.1.1 DNA restriction digest 42 2.1.2 Agarose gel electrophoresis 42 2.1.3 DNA fragment isolation 43 2.1.4 DNA fragment ligation 43 2.1.5 Transformation of competent E.Coli cells 43 2.1.6 Isolation of plasmid DNA 43 2.1.7 Measurement of DNA concentration 44 2.1.8 Plasmid DNA sequencing 44 2.1.9 Expression and purification of recombinant proteins 44 2.1.10 PCR– polymerase chain reaction 44 2.1.11 Quantitative PCR 46 2.1.11.1 RNA isolation and reverse transcription 46 2.1.11.2 qPCR 46 2.1.12 Site-directed mutagenesis 47 2.1.13 Genomic DNA isolation 47 2.2 Cell biology methods 48 2.2.1 Cell culture 48 2.2.2 Cell cryopreservation 48 2.2.3 Cell stimulation 49 2.2.4 Transient transfection of DNA 49 2.2.5 Stable transfection of DNA 49 2.2.5.1 Flp-InTM system 49 2.2.5.2 Flp-InTM T-RexTM system 50 2.2.5.3 pMOWS system 50 2.2.6 Total cell protein extractions 50 2.2.7 Subcellular fractionation 51 2.2.8 Measurement of protein concentration 52 2.3 Biochemistry methods 52 2.3.1 SDS-Polyacrylamide-gel electrophoresis (SDS-PAGE) 52 2.3.2 Immunoblotting (Western-blot) 54 2.3.3 Coimmunoprecipitation 55 2.3.4 GST-pulldown 56 2.3.5 Electrophoretic mobility shift assay 56 2.4 Confocal laser scanning microscopy methods 58 2.4.1 Description 58 2.4.2 Microscope settings 58 2.4.3 Indirect immunofluorescence staining of cells 59 2.4.4 Live cell imaging 60 2.4.5 Quantification of nuclear STAT3-FP amounts 60 III. Results 61 1. Characterization of stably transfected MEF ∆/∆ cells 61 2.The role of N-terminal domain and GAS-site recognition in STAT3 signaling 65 2.1 Ligand-induced nuclear accumulation and DNA-binding ability of STAT3 are independent of each other. 65 2.2 STAT3 N-terminal domain deletion mutant remains in the cytoplasm in the form of activated dimers capable of GAS-element binding 68 2.3 STAT3 NTD is not required for binding to various importin-α isoforms 71 2.4 Nuclear export inhibition does not rescue impaired nuclear accumulation of (∆N)STAT3 75 2.5 Basal nucleocytoplasmic shuttling does not require functional N-, SH2 or C-terminal domains 79 3. STAT3-mediated regulation of STAT1 signaling 81 3.1 Mutual intracellular crossregulation between STAT1 and STAT3 is asymmetric. 81 3.2 STAT3-mediated downregulation of IL-6-induced STAT1 activation relies on STAT3 transcriptional activity, but not on unique NTD functions. 84 4. The crosstalk between NF-κB subunit p65 and STAT3 89 4.1 Simultaneous visualization of p65 and STAT3 requires combined PFA and methanol treatment. 89 4.2 Absence of STAT3 has no significant effect on canonical TNFα- induced NF-κB activation, nuclear translocation and target gene expression. 91 4.3 Absence of NF-κB p65 subunit has no influence on IL-6-induced STAT3 signaling but leads to a decrease in STAT3 and STAT1 levels. 93 4.4 Inducible overexpression of STAT3 does not alter NF-κB signaling, while total p65 increase leads to increased IL-6 induced STAT3 signaling and atypical STAT1 nuclear accumulation. 97 4.5 Weak interaction between NF-κB p65 and STAT3 could be detected after TNFα, but not after IL-6 treatment. 100 5. Characterization of STAT3-YFP knock-in mice. 101 5.1 Generation and validation of transgenic mice. 101 5.2 Characterization of transgenic mice. 102 IV. Discussion 104 1. STAT3-FP reproduces functional characteristics of endogenous STAT3 105 2. GAS‐element‐specific DNA recognition is dispensable for nuclear accumulation of STAT3 106 3. STAT3 N-terminal domain deletion mutant remains in the cytoplasm in the form of activated dimers capable of GAS-element binding 108 4. STAT3 NTD is not required for binding to various importin-α isoforms 110 5. Latent nucleocytoplasmic shuttling of STAT3 does neither require GAS-element recognition, nor functional N-terminal or SH2 domains 113 6. STAT3 downregulates gp130/STAT1 signaling via target gene expression 116 7. Canonical signaling of STAT3 and NF-κB are independent of each other but NF-κB supports expression and activation of STAT1 and STAT3 122 8. STAT3-YFP knock-in mice have been successfully generated 127 V. Summary and Outlook 129 VI. References 135 VII. Abbreviations 156 Acknowledgements 165 Curriculum Vitae (Deutsch) 167 Curriculum Vitae (English) 169 Eidesstattliche Erklärung 171 „Research is what I'm doing when I don't know what I'm doing.” Wernher von Braun „A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.” Marie Curie „The human brain is an incredible pattern-matching machine.” Jeff Bezos Publications and coauthorships Essential parts of this thesis are presented in following publications: Martincuks A, Fahrenkamp D, Haan S, Herrmann A, Küster A, Müller-Newen G. Dissecting functions of the N-terminal domain and GAS-site recognition in STAT3 nuclear trafficking. Cell Signal. 2016, 28(8):810-25. Martincuks A, Andryka K, Küster A, Schmitz-Van de Leur H, Komorowski M, Müller-Newen G. Nuclear translocation of STAT3 and NF-κB are independent of each other but NF-B supports expression and activation of STAT3. Cell Signal. 2017, 32:36-47. Martincuks A, Küster A, Schmitz-Van de Leur H, Müller-Newen G. STAT3- mediated regulation of gp130/STAT1 signaling relies on STAT3 transcriptional activity. (Manuscript in preparation) Further publications: Domoszlai T, Martincuks A, Fahrenkamp D, Schmitz-Van de Leur H, Küster A, Müller-Newen G. Consequences of the disease-related L78R mutation for dimerization and activity of STAT3. J Cell Sci. 2014, 127(Pt 9):1899-910. Schumacher A, Denecke B, Braunschweig T, Stahlschmidt J, Ziegler S, Brandenburg LO, Stope MB, Martincuks A, Vogt M, Görtz D, Camporeale A, Poli V, Müller-Newen G, Brümmendorf TH, Ziegler P. Angptl4 is upregulated under inflammatory conditions in the bone marrow of mice, expands myeloid progenitors, and accelerates reconstitution of platelets after myelosuppressive therapy. J Hematol Oncol. 2015, 8:64. 2 Martin L, Peters C, Schmitz S, Moellmann J, Martincuks A, Heussen N, Lehrke M, Müller-Newen G, Marx G, Schuerholz T. Soluble Heparan Sulfate in Serum of Septic Shock Patients Induces Mitochondrial Dysfunction in Murine Cardiomyocytes. Shock. 2015, 44(6):569-77. Martin L, Peters C, Heinbockel L, Moellmann J, Martincuks A, Brandenburg K, Lehrke M, Müller-Newen G, Marx G, Schuerholz T. The synthetic antimicrobial peptide 19-2.5 attenuates mitochondrial dysfunction in cardiomyocytes stimulated with human sepsis serum. Innate Immunity. (Manuscript in revision) 3 Zusammenfassung STAT3 (signal transducer and activator of transcription 3) ist ein ubiquitärer Transkriptionsfaktor, der in vielen biologischen Prozessen, wie Hämatopoese, Entwicklung und Immunantwort involviert ist. Eine Dysregulation der STAT3- Signaltransduktion ist bei der Enstehung und Progression von chronischen Entzündungen, Krebserkrankungen und Fibrose beteiligt. Der erste Teil dieser Arbeit befasst sich mit den Funktionen der N-terminale Domäne (NTD) von STAT3 und der spezifischen DNS Bindung an GAS-Elemente bei der IL-6 vermittelten STAT3 Signalübertragung.