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The phosphoproteome of toll-like receptor-activated macrophages.

Weintz G, Olsen JV, Frühauf K, Niedzielska M, Amit I, Jantsch J, Mages J, Frech C, Dölken L, Mann M, Lang R - Mol. Syst. Biol. (2010)

Bottom Line: 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).Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany.

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.

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The phosphoproteome of LPS-activated macrophages—overview. Hotspots of TLR4-induced phosphorylation at the level of pathways, kinases and biological processes are summarised. Various signalling modules, cytoskeletal re-arrangement, cell cycle proteins and the translation machinery use the reversible protein modification for controlled activation that is both rapid and transient, as accomplished by phosphatase activity and phosphoprotein degradation. Most importantly, changes in phosphorylation activate TFs followed by inflammatory gene transcription indispensable for host defence. Selected examples of phosphoproteins are shown for each process. Depicted TFs have enriched evolutionary conserved binding sites in the promoters of highly induced LPS-target genes or were experimentally validated. Asterisks indicate LPS-regulated phosphorylation on a protein or kinase target.
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f4: The phosphoproteome of LPS-activated macrophages—overview. Hotspots of TLR4-induced phosphorylation at the level of pathways, kinases and biological processes are summarised. Various signalling modules, cytoskeletal re-arrangement, cell cycle proteins and the translation machinery use the reversible protein modification for controlled activation that is both rapid and transient, as accomplished by phosphatase activity and phosphoprotein degradation. Most importantly, changes in phosphorylation activate TFs followed by inflammatory gene transcription indispensable for host defence. Selected examples of phosphoproteins are shown for each process. Depicted TFs have enriched evolutionary conserved binding sites in the promoters of highly induced LPS-target genes or were experimentally validated. Asterisks indicate LPS-regulated phosphorylation on a protein or kinase target.

Mentions: We identified 48 phosphoproteins annotated as members of the murine or human TLR, MAPK or NFKB signalling pathways in InnateDB or on the innate immunity signalling poster compiled by Latz and Fitzgerald (2008), 31 of which showed LPS-regulated phosphorylation (Supplementary Table S2). The pathway annotation ‘TLR signalling' showed a trend for enrichment among LPS-regulated phosphoproteins compared to non-regulated phosphoproteins (odds ratio 2.4; P-value 0.15). Significant over-representation was found for MAPK signalling members and pathways recently described as activated downstream of TLRs, for example, the AKT and mTOR pathways and the Rho GTPase cycle (Ruse and Knaus, 2006) (Table IIA; Supplementary Figure S5). GO analysis showed enrichment of the terms ‘signal transduction', ‘cell communication' and ‘kinase activity' (Table IIB; Supplementary Table S3). Interestingly, functional annotation terms associated with the cytoskeleton were also significantly enriched among LPS-regulated phosphoproteins. ‘Cell proliferation' showed a trend for over-representation (odds ratio 4.6; corrected P-value 0.12), consistent with over-representation of motifs for cell cycle-associated kinases observed above. In summary, unbiased statistical analyses of kinase motifs, signalling pathways and functional GO annotation highlighted known and novel players of TLR signalling and linked TLR activation to the cytoskeleton and cell proliferation (see overview in Figure 4).


The phosphoproteome of toll-like receptor-activated macrophages.

Weintz G, Olsen JV, Frühauf K, Niedzielska M, Amit I, Jantsch J, Mages J, Frech C, Dölken L, Mann M, Lang R - Mol. Syst. Biol. (2010)

The phosphoproteome of LPS-activated macrophages—overview. Hotspots of TLR4-induced phosphorylation at the level of pathways, kinases and biological processes are summarised. Various signalling modules, cytoskeletal re-arrangement, cell cycle proteins and the translation machinery use the reversible protein modification for controlled activation that is both rapid and transient, as accomplished by phosphatase activity and phosphoprotein degradation. Most importantly, changes in phosphorylation activate TFs followed by inflammatory gene transcription indispensable for host defence. Selected examples of phosphoproteins are shown for each process. Depicted TFs have enriched evolutionary conserved binding sites in the promoters of highly induced LPS-target genes or were experimentally validated. Asterisks indicate LPS-regulated phosphorylation on a protein or kinase target.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2913394&req=5

f4: The phosphoproteome of LPS-activated macrophages—overview. Hotspots of TLR4-induced phosphorylation at the level of pathways, kinases and biological processes are summarised. Various signalling modules, cytoskeletal re-arrangement, cell cycle proteins and the translation machinery use the reversible protein modification for controlled activation that is both rapid and transient, as accomplished by phosphatase activity and phosphoprotein degradation. Most importantly, changes in phosphorylation activate TFs followed by inflammatory gene transcription indispensable for host defence. Selected examples of phosphoproteins are shown for each process. Depicted TFs have enriched evolutionary conserved binding sites in the promoters of highly induced LPS-target genes or were experimentally validated. Asterisks indicate LPS-regulated phosphorylation on a protein or kinase target.
Mentions: We identified 48 phosphoproteins annotated as members of the murine or human TLR, MAPK or NFKB signalling pathways in InnateDB or on the innate immunity signalling poster compiled by Latz and Fitzgerald (2008), 31 of which showed LPS-regulated phosphorylation (Supplementary Table S2). The pathway annotation ‘TLR signalling' showed a trend for enrichment among LPS-regulated phosphoproteins compared to non-regulated phosphoproteins (odds ratio 2.4; P-value 0.15). Significant over-representation was found for MAPK signalling members and pathways recently described as activated downstream of TLRs, for example, the AKT and mTOR pathways and the Rho GTPase cycle (Ruse and Knaus, 2006) (Table IIA; Supplementary Figure S5). GO analysis showed enrichment of the terms ‘signal transduction', ‘cell communication' and ‘kinase activity' (Table IIB; Supplementary Table S3). Interestingly, functional annotation terms associated with the cytoskeleton were also significantly enriched among LPS-regulated phosphoproteins. ‘Cell proliferation' showed a trend for over-representation (odds ratio 4.6; corrected P-value 0.12), consistent with over-representation of motifs for cell cycle-associated kinases observed above. In summary, unbiased statistical analyses of kinase motifs, signalling pathways and functional GO annotation highlighted known and novel players of TLR signalling and linked TLR activation to the cytoskeleton and cell proliferation (see overview in Figure 4).

Bottom Line: 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).Finally, weaving together phosphoproteome and nascent transcriptome data by in silico promoter analysis, we implicated several phosphorylated TFs in primary LPS-controlled gene expression.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany.

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.

Show MeSH
Related in: MedlinePlus