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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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Pharmacological inhibition of LPS-activated pathways differentially impacts gene expression. (A) Induction of selected LPS-target genes. Strongly LPS-induced genes were identified by microarray analysis of nascent RNA (see Figure 6). The changes in expression of 10 selected target genes were analysed by qRT–PCR with Roche Universal Probe Library reagents in independent experiments using total RNA from macrophages generated by the standard protocol after 45 min and 4.5 h. Shown are mean and s.d. of quadruplicate PCR results from a representative experiment of two performed. (B) Effects of pharmacological inhibitors on LPS-induced gene expression. Macrophages were treated and analysed as in (A); the pharmacological inhibitors were added 2 h before stimulation with LPS. Fold-changes induced by LPS were calculated relative to the untreated samples using the ΔΔCT method and subsequently normalised to the effect of LPS in the absence of inhibitor. The experiment was performed two times. For visualisation of the inhibitor effects, the data were colour coded to indicate inhibition (blue) or increased induction (red) by a factor of >2 in both (dark colour) or one of the experiments (light colour). (C) ATM-inhibition increases expression of IL-10, CCL2 and CXCL10. Macrophages were pretreated with different concentrations of ATM inhibitor (μM) or solvent (DMSO) for 2 h, followed by stimulation with titrated amounts of LPS for 4.5 h. qRT–PCR data of quadruplicate samples from one representative experiment of two to three performed are shown. (D) Increased ATM substrate phosphorylation after LPS is blocked by ATM inhibitor. After pretreatment with DMSO or ATM inhibitor (10 μM) for 2 h, macrophages were stimulated for 1 h with LPS as indicated. Phosphorylated ATM substrate proteins were detected with Cell Signaling antibody Cat. #2851.
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f5: Pharmacological inhibition of LPS-activated pathways differentially impacts gene expression. (A) Induction of selected LPS-target genes. Strongly LPS-induced genes were identified by microarray analysis of nascent RNA (see Figure 6). The changes in expression of 10 selected target genes were analysed by qRT–PCR with Roche Universal Probe Library reagents in independent experiments using total RNA from macrophages generated by the standard protocol after 45 min and 4.5 h. Shown are mean and s.d. of quadruplicate PCR results from a representative experiment of two performed. (B) Effects of pharmacological inhibitors on LPS-induced gene expression. Macrophages were treated and analysed as in (A); the pharmacological inhibitors were added 2 h before stimulation with LPS. Fold-changes induced by LPS were calculated relative to the untreated samples using the ΔΔCT method and subsequently normalised to the effect of LPS in the absence of inhibitor. The experiment was performed two times. For visualisation of the inhibitor effects, the data were colour coded to indicate inhibition (blue) or increased induction (red) by a factor of >2 in both (dark colour) or one of the experiments (light colour). (C) ATM-inhibition increases expression of IL-10, CCL2 and CXCL10. Macrophages were pretreated with different concentrations of ATM inhibitor (μM) or solvent (DMSO) for 2 h, followed by stimulation with titrated amounts of LPS for 4.5 h. qRT–PCR data of quadruplicate samples from one representative experiment of two to three performed are shown. (D) Increased ATM substrate phosphorylation after LPS is blocked by ATM inhibitor. After pretreatment with DMSO or ATM inhibitor (10 μM) for 2 h, macrophages were stimulated for 1 h with LPS as indicated. Phosphorylated ATM substrate proteins were detected with Cell Signaling antibody Cat. #2851.

Mentions: In summary, this study provides a novel and global perspective on innate immune activation by TLR signalling (Figure 5). We quantitatively detected a large number of previously unknown site-specific phosphorylation events, which are now publicly available through the Phosida database. By combining different data mining approaches, we consistently identified canonical and newly implicated TLR-activated signalling modules. In particular, the PI3K/AKT and the related mTOR pathway were highlighted; furthermore, DNA damage–response associated ATM/ATR kinases and the cytoskeleton emerged as unexpected hotspots for phosphorylation. Finally, weaving together corresponding phophoproteome and nascent transcriptome datasets through the loom of in silico promoter analysis we identified TFs with a likely role in mediating TLR-induced gene expression programmes.


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)

Pharmacological inhibition of LPS-activated pathways differentially impacts gene expression. (A) Induction of selected LPS-target genes. Strongly LPS-induced genes were identified by microarray analysis of nascent RNA (see Figure 6). The changes in expression of 10 selected target genes were analysed by qRT–PCR with Roche Universal Probe Library reagents in independent experiments using total RNA from macrophages generated by the standard protocol after 45 min and 4.5 h. Shown are mean and s.d. of quadruplicate PCR results from a representative experiment of two performed. (B) Effects of pharmacological inhibitors on LPS-induced gene expression. Macrophages were treated and analysed as in (A); the pharmacological inhibitors were added 2 h before stimulation with LPS. Fold-changes induced by LPS were calculated relative to the untreated samples using the ΔΔCT method and subsequently normalised to the effect of LPS in the absence of inhibitor. The experiment was performed two times. For visualisation of the inhibitor effects, the data were colour coded to indicate inhibition (blue) or increased induction (red) by a factor of >2 in both (dark colour) or one of the experiments (light colour). (C) ATM-inhibition increases expression of IL-10, CCL2 and CXCL10. Macrophages were pretreated with different concentrations of ATM inhibitor (μM) or solvent (DMSO) for 2 h, followed by stimulation with titrated amounts of LPS for 4.5 h. qRT–PCR data of quadruplicate samples from one representative experiment of two to three performed are shown. (D) Increased ATM substrate phosphorylation after LPS is blocked by ATM inhibitor. After pretreatment with DMSO or ATM inhibitor (10 μM) for 2 h, macrophages were stimulated for 1 h with LPS as indicated. Phosphorylated ATM substrate proteins were detected with Cell Signaling antibody Cat. #2851.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Pharmacological inhibition of LPS-activated pathways differentially impacts gene expression. (A) Induction of selected LPS-target genes. Strongly LPS-induced genes were identified by microarray analysis of nascent RNA (see Figure 6). The changes in expression of 10 selected target genes were analysed by qRT–PCR with Roche Universal Probe Library reagents in independent experiments using total RNA from macrophages generated by the standard protocol after 45 min and 4.5 h. Shown are mean and s.d. of quadruplicate PCR results from a representative experiment of two performed. (B) Effects of pharmacological inhibitors on LPS-induced gene expression. Macrophages were treated and analysed as in (A); the pharmacological inhibitors were added 2 h before stimulation with LPS. Fold-changes induced by LPS were calculated relative to the untreated samples using the ΔΔCT method and subsequently normalised to the effect of LPS in the absence of inhibitor. The experiment was performed two times. For visualisation of the inhibitor effects, the data were colour coded to indicate inhibition (blue) or increased induction (red) by a factor of >2 in both (dark colour) or one of the experiments (light colour). (C) ATM-inhibition increases expression of IL-10, CCL2 and CXCL10. Macrophages were pretreated with different concentrations of ATM inhibitor (μM) or solvent (DMSO) for 2 h, followed by stimulation with titrated amounts of LPS for 4.5 h. qRT–PCR data of quadruplicate samples from one representative experiment of two to three performed are shown. (D) Increased ATM substrate phosphorylation after LPS is blocked by ATM inhibitor. After pretreatment with DMSO or ATM inhibitor (10 μM) for 2 h, macrophages were stimulated for 1 h with LPS as indicated. Phosphorylated ATM substrate proteins were detected with Cell Signaling antibody Cat. #2851.
Mentions: In summary, this study provides a novel and global perspective on innate immune activation by TLR signalling (Figure 5). We quantitatively detected a large number of previously unknown site-specific phosphorylation events, which are now publicly available through the Phosida database. By combining different data mining approaches, we consistently identified canonical and newly implicated TLR-activated signalling modules. In particular, the PI3K/AKT and the related mTOR pathway were highlighted; furthermore, DNA damage–response associated ATM/ATR kinases and the cytoskeleton emerged as unexpected hotspots for phosphorylation. Finally, weaving together corresponding phophoproteome and nascent transcriptome datasets through the loom of in silico promoter analysis we identified TFs with a likely role in mediating TLR-induced gene expression programmes.

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