The TAK1 and canonical IKK complexes are the two master protein kinases of the innate immune system that control the production of inflammatory mediators, but the mechanisms by which they are activated in this system are still unclear. In this thesis, I present the research I have carried out to solve these problems. The IKKb component of the canonical IKK complex is required to activate the transcription factors NF-kB and IRF5 and the protein kinase Tpl2, but how IKKβ itself is activated in vivo is still unclear. It was found to require phosphorylation by one or more ‘upstream’ protein kinases in some reports, but by autophosphorylation in others. In the first part of this thesis, I describe my work that has resolved this controversy by demonstrating that the activation of IKKb induced by IL-1 (interleukin-1) or TNF (tumour necrosis factor) in embryonic fibroblasts, or by ligands that activate Toll-like receptors in macrophages, requires two distinct phosphorylation events: first, the TAK1 catalysed phosphorylation of Ser177 and, secondly, the IKKb-catalysed autophosphorylation of Ser181. The phosphorylation of Ser177 by TAK1 is a priming event required for the subsequent autophosphorylation of Ser181, which enables IKKk to phosphorylate exogenous substrates. I also present genetic evidence which indicates that the IL-1-stimulated, LUBAC (linear ubiquitin chain assembly complex)-catalysed formation of Met1-linked/linear ubiquitin (Met1-Ub) chains and their interaction with the NEMO (NF-kB essential modulator) component of the canonical IKK complex permits the TAK1-catalysed priming phosphorylation of IKKb at Ser177 and IKKa at Ser176. These findings may be of general significance for the activation of other protein kinases. The activation of the TAK1 complex by inflammatory stimuli is thought to be triggered by the binding of Lys63-linked ubiquitin chains to the TAB2 or TAB3 components of the TAB1-TAK1-TAB2 and TAB1-TAK1-TAB3 complexes. In the second part of the thesis I tested whether this broadly accepted model was correct by knocking out the genes encoding TAK1 and its regulatory subunits TAB1, TAB2 and TAB3 by CRISPR/Cas9 gene-editing technology, alone and in combination, in an IL-1 receptor expressing human cell line. These genetic studies led me to discover that the IL-1-dependent activation of TAK1 occurs by two different mechanisms. The first, involves the previously described interaction of Lys63-linked ubiquitin chains with TAB2 and TAB3, while the second can take place in the complete absence of TAB2 and TAB3. The second mechanism, which involves activation of the TAB1-TAK1 heterodimer is more transient than the first, but is sufficient for the IL-1-dependent transcription of immediate early genes (A20, IkBa). I show that the activation of the TAB1-TAK1 complex requires the expression of the E3 ubiquitin ligase TRAF6 and the TRAF6-generated formation of Lys63-linked ubiquitin chains, which leads to the phosphorylation of TAK1 at Thr187 and activation. However, neither TAB1 nor TAK1 bind directly to Lys63-linked ubiquitin chains. I identify one novel IL-1-dependent phosphorylation site on TAB1 and two on TAK1 and propose that Lys63-linked ubiquitin chains activate an as yet unidentified protein kinase, which phosphorylates one or more of the novel phosphorylation sites on the TAB1-TAK1 heterodimer inducing a conformational change that permits TAK1 to autophosphorylate Thr187.

Bisher wurde davon ausgegangen, dass der IKK-komplex durch Regulation des Transkriptionsfaktors NF-kappaB die stressinduzierte Expression von Zielgenen steuert. Im Rahmen der hier vorgelegten Dissertation konnte jedoch gezeigt werden, dass der IKK-Komplex unabhängig von seiner Rolle in der NF-kappaB-Aktivierung die Stabilität einer Vielzahl von mRNAs kontrolliert. Mittels der Kombination von Ko-Immunopräzipitationsstudien und SILAC-MS konnte die induzierte Interaktion der regulatorischen Untereinheit des IKK-Komplexes IKKgamma mit dem Gerüstprotein EDC4 (Enhancer of Decapping 4) nachgewiesen werden. EDC4 ist eine essentielle Komponente sogenannter zytoplasmatischer „Processing Bodies“ (P-Bodies). Diese fungieren als Depots für die Speicherung von mRNAs, aber auch als Orte der mRNA-Degradation und der miRNA-vermittelten Repression spezifischer Zielgene. Die Interaktion von IKKgamma mit EDC4 konnte durch verschiedene Stimuli induziert werden. Dazu zählen DNA-Schäden durch Doppelstrangbrüche, aber auch die Aktivierung von Oberflächenrezeptoren durch TNFalpha und IL-1beta. EDC4 dient darüber hinaus als Substrat der Kinase IKKbeta. Mittels Massenspektrometrie und Kinaseassays konnten vier IKK-abhängige Phosphorylierungsstellen identifiziert werden. Die IKK-vermittelte Phosphorylierung von EDC4 ist essentiell für die Regulation von mRNAs und die damit verbundene Bildung der zytoplasmatischen P-Bodies. Diese Befunde konnten sowohl in stabilen induzierbaren Zelllinien, mittels transienter Transfektion und durch den Gebrauch von Kinaseinhibitoren in primären als auch in Krebszelllinien bestätigt werden. mRNA-Stabilitätsassays und eine RNA-Seq Analyse bestätigten die stressinduzierten Änderungen in den Halbwertszeiten spezifischer Transkripte und offenbarten einen gemeinsamen Regulationsmechanismus des IKK-Komplexes mit EDC4. / The IKK complex is deemed to regulate gene expression through the activation of the transcription factor NF-kappaB. Here I describe an NF-kappaB-independent function of the IKK complex in regulating mRNA stability across different cell types and stimuli. A SILAC-MS screen for interaction partners of the regulatory subunit IKKgamma revealed an inducible interaction with Enhancer of mRNA Decapping 4 (EDC4). EDC4 is an essential component of cytoplasmic processing bodies (P-bodies). P-bodies function as sites of mRNA storage, degradation and miRNA-mediated silencing. Interaction between IKKgamma and EDC4 can be induced by various stimuli, including DNA damage, TNFalpha and IL-1beta. EDC4 was identified as a novel IKK substrate and four IKKbeta phosphorylation sites were determined by mass spectrometry and in kinase assays. Stable inducible cell lines, transient transfection and kinase inhibitors were used in different human cancer and in primary cell lines and demonstrated that phosphorylation of EDC4 by IKK is essential for formation of P-Bodies in response to numerous stimuli. mRNA stability assays confirmed stress-induced changes in the half-life of target mRNAs and revealed common regulation of mRNA stability by IKK and EDC4. The transcriptome-wide reach of this joint regulation was assessed via RNA-Seq analysis.