The phosphoproteome of toll-like receptor-activated macrophages

First global and quantitative analysis of phosphorylation cascades induced by toll-like receptor (TLR) stimulation in macrophages identifies nearly 7000 phosphorylation sites and shows extensive and dynamic up-regulation and down-regulation after lipopolysaccharide (LPS).In addition to the canonical TLR-associated pathways, mining of the phosphorylation data suggests an involvement of ATM/ATR kinases in signalling and shows that the cytoskeleton is a hotspot of TLR-induced phosphorylation.Intersecting transcription factor phosphorylation with bioinformatic promoter analysis of genes induced by LPS identified several candidate transcriptional regulators that were previously not implicated in TLR-induced transcriptional control

Phosphoproteom-Analyse der Makrophagenantwort auf Toll-like Rezeptor (TLR)-Aktivierung

Macrophages are the immune system’s first line of defence. Via pattern recognition receptors microbes are recognised as foreign and elicit a rapid massive production of cytokines, chemokines and other mediators important for host defence. Recognition of bacterial lipopolysaccharide (LPS) by Toll-like receptor 4 (TLR4) activates some known signalling pathways (MAPK, NF-κB) and transcription factors (CREB/AP-1, NF-κB, IRF). Through a phosphoproteome analysis, this thesis provides for the first time a global and quantitative picture of the involved signalling pathways, kinases and transcription factors. Bioinformatic analyses for enriched kinase motifs, Gene Ontology and signalling pathway annotation identified in particular the PI3K/AKT, mTOR and Ca2+ pathways, as well as the cell cycle and the cytoskeleton as novel hotspots of LPS-regulated phosphorylation. Finally, integration of phosphoproteome and nascent transcriptome data by in silico promoter analysis identified novel transcription factors, which act at the intersection of TLR-induced kinase activation and gene expression.Makrophagen sind die erste Alarm-Zentrale des Immunsystems. Über Mustererkennungsrezeptoren werden Pathogene als fremd erkannt und eine schnelle massive Produktion von Zytokinen, Chemokinen und anderen Mediatoren zur Erregerabwehr ausgelöst. Die Erkennung bakteriellen Lipopolysaccharids (LPS) durch Toll-like Rezeptor 4 (TLR4) aktiviert einige bekannte Signalwege (MAPK, NF-κB) und Transkriptionsfaktoren (TFs) (CREB/AP-1, NF-κB, IRF). Diese Arbeit liefert durch eine Phosphoproteom-Analyse erstmals ein globales quantitatives Bild der beteiligten Signalwege, Kinasen und TFs. Bioinformatische Analysen für angereicherte Kinase-Motive, Gene Ontology- und Signalweg-Annotation identifizierten insbesondere Wege über PI3K/AKT, mTOR und Ca2+, sowie Zellzyklus und Zytoskelett als neue Module mit LPS-regulierter Phosphorylierung. Durch Integration der Phosphoproteom- mit Transkriptomdaten mittels in silico Promotor-Analyse konnten außerdem neue TFs identifiziert werden, die zwischen TLR-induzierter Kinaseaktivierung und Genexpression vermitteln