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.
| Original language | English |
|---|---|
| Journal | Molecular Systems Biology |
| Volume | 6 |
| Pages (from-to) | 371 |
| ISSN | 1744-4292 |
| DOIs | |
| Publication status | Published - 8 Jun 2010 |
Keywords
- Animals
- Cells, Cultured
- Enzyme Activation
- Lipopolysaccharides
- Macrophage Activation
- Macrophages
- Mice
- Phosphoproteins
- Phosphorylation
- Protein Kinases
- Proteome
- Signal Transduction
- Toll-Like Receptor 4
- Transcription Factors
- Transcriptional Activation