The phosphoproteome of toll-like receptor-activated macrophages

Molecular Systems Biology 6: 371; published online 8 June 2010; doi:10.1038/msb.2010.29

The phosphoproteome of toll-like receptor-activated macrophages

Gabriele Weintz1,2,8, Jesper V Olsen3,4,8, Katja Fruehauf1, Magdalena Niedzielska2, Ido Amit5, Jonathan Jantsch2, Joerg Mages1, Cornelie Frech6, Lars Doelken7, Matthias Mann3 and Roland Lang1,2,*

1 Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany, 2 Microbiological Institute–Clinical Microbiology, Immunology and Hygiene, University Clinic of Erlangen, Erlangen, Germany, 3 Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Martinsried, Germany, 4 Department of Proteomics, Faculty of Health Sciences, Novo Nordisk Foundation Center for Protein Research, University of Copenhagen, Copenhagen, Denmark, 5 Broad Institute of Harvard and MIT, Boston, MA, USA, 6 Genomatix, Munich, Germany and 7 Max von Pettenkofer-Institute, Ludwig Maximilians University Munich, Munich, Germany 8 These authors contributed equally to this work * Corresponding author. Clinical Microbiology, Immunology and Hygiene, University Clinics Erlangen, Wasserturmstr. 3-5, Erlangen 91054, Germany.

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.

http://www.nature.com/msb/journal/v6/n1/full/msb201029.html