TY - JOUR

T1 - The phosphoproteome of toll-like receptor-activated macrophages

AU - Weintz, Gabriele

AU - Olsen, Jesper Velgaard

AU - Frühauf, Katja

AU - Niedzielska, Magdalena

AU - Amit, Ido

AU - Jantsch, Jonathan

AU - Mages, Jörg

AU - Frech, Cornelie

AU - Dölken, Lars

AU - Mann, Matthias

AU - Lang, Roland

PY - 2010/6/8

Y1 - 2010/6/8

N2 - 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.

AB - 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.

KW - Animals

KW - Cells, Cultured

KW - Enzyme Activation

KW - Lipopolysaccharides

KW - Macrophage Activation

KW - Macrophages

KW - Mice

KW - Phosphoproteins

KW - Phosphorylation

KW - Protein Kinases

KW - Proteome

KW - Signal Transduction

KW - Toll-Like Receptor 4

KW - Transcription Factors

KW - Transcriptional Activation

U2 - 10.1038/msb.2010.29

DO - 10.1038/msb.2010.29

M3 - Journal article

C2 - 20531401

VL - 6

SP - 371

JO - Molecular Systems Biology

JF - Molecular Systems Biology

SN - 1744-4292

ER -