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

Amit, Ido

Dölken, Lars

Frech, Cornelie

Frühauf, Katja

Jantsch, Jonathan

Lang, Roland

Mages, Jörg

Mann, Matthias

Niedzielska, Magdalena

Olsen, Jesper V

Weintz, Gabriele

Molecular Systems Biology

English

PubMed

Olsen, Jesper Velgaard

Copenhagen University Research Information System

The phosphoproteome of toll-like receptor-activated macrophages

Recognition of microbial danger signals by toll‐like receptors (TLR) causes re‐programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome of primary macrophages using stable isotope labelling with amino acids in cell culture, phosphopeptide enrichment and high‐resolution mass spectrometry. In parallel, nascent RNA was profiled to link transcription factor (TF) phosphorylation to TLR4‐induced transcriptional activation. We reproducibly identified 1850 phosphoproteins with 6956 phosphorylation sites, two thirds of which were not reported earlier. LPS caused major dynamic changes in the phosphoproteome (24% up‐regulation and 9% down‐regulation). Functional bioinformatic analyses confirmed canonical players of the TLR pathway and highlighted other signalling modules (e.g. mTOR, ATM/ATR kinases) and the cytoskeleton as hotspots of LPS‐regulated phosphorylation. Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS‐controlled gene expression

Gabriele Weintz

Jesper V Olsen

Katja Frühauf

Magdalena Niedzielska

Ido Amit

Jonathan Jantsch

Jörg Mages

Cornelie Frech

Lars Dölken

Matthias Mann

Roland Lang

Directory of Open Access Journals

The phosphoproteome of toll‐like receptor‐activated macrophages

Weintz, G.

Olsen, J. V.

Frühauf, K.

Niedzielska, M.

Amit, I.

Jantsch, J.

Mages, J.

Frech, C.

Dölken, L.

Mann, M.

Lang, R.

Max Planck Society eDoc Server

Cell cycle checkpoint signaling through the ATM and ATR kinases.

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IRFs: master regulators of signalling by Toll-like receptors and cytosolic pattern-recognition receptors. Nat Rev Immunol 6: 644–658

STRING 8–a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res 37: D412–D416 Karin M

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The phosphoproteome of toll-like receptor-activated macrophages

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Copenhagen University Research Information System

Directory of Open Access Journals

Max Planck Society eDoc Server