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.
Weintz
Gabriele
G
Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany.
Olsen
Jesper V
JV
Frühauf
Katja
K
Niedzielska
Magdalena
M
Amit
Ido
I
Jantsch
Jonathan
J
Mages
Jörg
J
Frech
Cornelie
C
Dölken
Lars
L
Mann
Matthias
M
Lang
Roland
R
D000818:Animals; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation / Q000187:drug effects / Q000276:immunology*; D008264:Macrophages / Q000187:drug effects / Q000201:enzymology / Q000276:immunology*; D051379:Mice; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000276:immunology*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000235:genetics
D008070:Lipopolysaccharides; D010750:Phosphoproteins; D020543:Proteome; D051197:Toll-Like Receptor 4; D014157:Transcription Factors; D011494:Protein Kinases