The phosphoproteome of toll-like receptor-activated macrophages

Mol Syst Biol. 2010 Jun 8;6:371. doi: 10.1038/msb.2010.29.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Enzyme Activation / drug effects
  • Lipopolysaccharides / pharmacology
  • Macrophage Activation / drug effects
  • Macrophage Activation / immunology*
  • Macrophages / drug effects
  • Macrophages / enzymology
  • Macrophages / immunology*
  • Mice
  • Phosphoproteins / metabolism*
  • Phosphorylation / drug effects
  • Protein Kinases / metabolism
  • Proteome / metabolism*
  • Signal Transduction / drug effects
  • Toll-Like Receptor 4 / immunology*
  • Transcription Factors / metabolism
  • Transcriptional Activation / drug effects
  • Transcriptional Activation / genetics

Substances

  • Lipopolysaccharides
  • Phosphoproteins
  • Proteome
  • Toll-Like Receptor 4
  • Transcription Factors
  • Protein Kinases