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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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LPS-induced TF phosphorylation and changes in the nascent transcriptome—in silico promoter analysis. (A) Phosphorylation sites on TFs. Phosphorylation sites up-regulated or down-regulated more than 1.5-fold in both experiments are indicated in grey. (B) Workflow for integration of phosphoproteome and transcriptome data. Microarray analyses of metabolically labelled nascent and of total cellular RNA were performed on macrophages. Promoter sequences of LPS-regulated genes (induction ⩾3-fold) and of genes that were not transcriptionally altered in response to LPS (2000 least regulated probe sets) were retrieved with Genomatix Gene2Promoter. Promoters were analysed for the presence of binding sites for all identified phosphorylated TF families with Genomatix RegionMiner, and significant over-representation in LPS-regulated promoters was determined (odds ratio ⩾1.3; corrected P-value ⩽0.05). (C–E) Increase in microarray sensitivity by analysis of nascent RNA. Microarray analysis of (C) nascent and (D) total cellular RNA from two independent experiments. Macrophages were left un-treated or stimulated with LPS for 45 min or 4.5 h. Nascent RNA was labelled by addition of 4sU during the last 35 min of stimulation and purified after extraction of total cellular RNA. For each comparison, the number of probe sets induced at least two-/three-/five-fold is represented in the upper left corner, the number of probe sets repressed at least two-/three-/five-fold in the lower right corner. (E) Venn diagrams illustrate the increased sensitivity for early changes in transcription for at least three-fold-induced probe sets of nascent compared to total RNA analyses. (F) TF families with over-represented binding sites in the promoters of transcriptionally LPS-regulated genes.
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f6: LPS-induced TF phosphorylation and changes in the nascent transcriptome—in silico promoter analysis. (A) Phosphorylation sites on TFs. Phosphorylation sites up-regulated or down-regulated more than 1.5-fold in both experiments are indicated in grey. (B) Workflow for integration of phosphoproteome and transcriptome data. Microarray analyses of metabolically labelled nascent and of total cellular RNA were performed on macrophages. Promoter sequences of LPS-regulated genes (induction ⩾3-fold) and of genes that were not transcriptionally altered in response to LPS (2000 least regulated probe sets) were retrieved with Genomatix Gene2Promoter. Promoters were analysed for the presence of binding sites for all identified phosphorylated TF families with Genomatix RegionMiner, and significant over-representation in LPS-regulated promoters was determined (odds ratio ⩾1.3; corrected P-value ⩽0.05). (C–E) Increase in microarray sensitivity by analysis of nascent RNA. Microarray analysis of (C) nascent and (D) total cellular RNA from two independent experiments. Macrophages were left un-treated or stimulated with LPS for 45 min or 4.5 h. Nascent RNA was labelled by addition of 4sU during the last 35 min of stimulation and purified after extraction of total cellular RNA. For each comparison, the number of probe sets induced at least two-/three-/five-fold is represented in the upper left corner, the number of probe sets repressed at least two-/three-/five-fold in the lower right corner. (E) Venn diagrams illustrate the increased sensitivity for early changes in transcription for at least three-fold-induced probe sets of nascent compared to total RNA analyses. (F) TF families with over-represented binding sites in the promoters of transcriptionally LPS-regulated genes.

Mentions: We were especially interested in the phosphorylation of TFs and its regulation by LPS (Figure 6A). We hypothesised that functionally important TFs should have an increased frequency of binding sites in the promoters of LPS-regulated genes (Figure 6B). To identify transcriptionally regulated genes with high sensitivity, we isolated nascent RNA after metabolic labelling (Figure 6C–E). In silico promoter scanning using Genomatix software for binding sites for all 50 TF families with phosphorylated members was used to test for enrichment in transciptionally induced genes (Figure 6F). At the early time point, binding site enrichment for the canonical TLR-associated TF NFkB was detected, and in addition we found that several other TF families with an established role in the transcription of individual LPS-target genes showed binding site enrichment (CEBP, MEF2, NFAT and HEAT). In addition, enrichment for OCT and HOXC binding sites at the early time point and SORY matrices later after stimulation indicated an involvement of the phosphorylated members of the respective TF families in the execution of TLR-induced transcriptional responses. An initial test of the function for a few of these candidate transcriptional regulators was performed using siRNA knockdown in primary macrophages. These experiments suggested that knock down of the SORY binding phosphoprotein Capicua homolog (Cic) and to a lesser extent of the CREB family member Atf7 selectively attenuates LPS-induced expression of Il1a and Il1b.


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)

LPS-induced TF phosphorylation and changes in the nascent transcriptome—in silico promoter analysis. (A) Phosphorylation sites on TFs. Phosphorylation sites up-regulated or down-regulated more than 1.5-fold in both experiments are indicated in grey. (B) Workflow for integration of phosphoproteome and transcriptome data. Microarray analyses of metabolically labelled nascent and of total cellular RNA were performed on macrophages. Promoter sequences of LPS-regulated genes (induction ⩾3-fold) and of genes that were not transcriptionally altered in response to LPS (2000 least regulated probe sets) were retrieved with Genomatix Gene2Promoter. Promoters were analysed for the presence of binding sites for all identified phosphorylated TF families with Genomatix RegionMiner, and significant over-representation in LPS-regulated promoters was determined (odds ratio ⩾1.3; corrected P-value ⩽0.05). (C–E) Increase in microarray sensitivity by analysis of nascent RNA. Microarray analysis of (C) nascent and (D) total cellular RNA from two independent experiments. Macrophages were left un-treated or stimulated with LPS for 45 min or 4.5 h. Nascent RNA was labelled by addition of 4sU during the last 35 min of stimulation and purified after extraction of total cellular RNA. For each comparison, the number of probe sets induced at least two-/three-/five-fold is represented in the upper left corner, the number of probe sets repressed at least two-/three-/five-fold in the lower right corner. (E) Venn diagrams illustrate the increased sensitivity for early changes in transcription for at least three-fold-induced probe sets of nascent compared to total RNA analyses. (F) TF families with over-represented binding sites in the promoters of transcriptionally LPS-regulated genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: LPS-induced TF phosphorylation and changes in the nascent transcriptome—in silico promoter analysis. (A) Phosphorylation sites on TFs. Phosphorylation sites up-regulated or down-regulated more than 1.5-fold in both experiments are indicated in grey. (B) Workflow for integration of phosphoproteome and transcriptome data. Microarray analyses of metabolically labelled nascent and of total cellular RNA were performed on macrophages. Promoter sequences of LPS-regulated genes (induction ⩾3-fold) and of genes that were not transcriptionally altered in response to LPS (2000 least regulated probe sets) were retrieved with Genomatix Gene2Promoter. Promoters were analysed for the presence of binding sites for all identified phosphorylated TF families with Genomatix RegionMiner, and significant over-representation in LPS-regulated promoters was determined (odds ratio ⩾1.3; corrected P-value ⩽0.05). (C–E) Increase in microarray sensitivity by analysis of nascent RNA. Microarray analysis of (C) nascent and (D) total cellular RNA from two independent experiments. Macrophages were left un-treated or stimulated with LPS for 45 min or 4.5 h. Nascent RNA was labelled by addition of 4sU during the last 35 min of stimulation and purified after extraction of total cellular RNA. For each comparison, the number of probe sets induced at least two-/three-/five-fold is represented in the upper left corner, the number of probe sets repressed at least two-/three-/five-fold in the lower right corner. (E) Venn diagrams illustrate the increased sensitivity for early changes in transcription for at least three-fold-induced probe sets of nascent compared to total RNA analyses. (F) TF families with over-represented binding sites in the promoters of transcriptionally LPS-regulated genes.
Mentions: We were especially interested in the phosphorylation of TFs and its regulation by LPS (Figure 6A). We hypothesised that functionally important TFs should have an increased frequency of binding sites in the promoters of LPS-regulated genes (Figure 6B). To identify transcriptionally regulated genes with high sensitivity, we isolated nascent RNA after metabolic labelling (Figure 6C–E). In silico promoter scanning using Genomatix software for binding sites for all 50 TF families with phosphorylated members was used to test for enrichment in transciptionally induced genes (Figure 6F). At the early time point, binding site enrichment for the canonical TLR-associated TF NFkB was detected, and in addition we found that several other TF families with an established role in the transcription of individual LPS-target genes showed binding site enrichment (CEBP, MEF2, NFAT and HEAT). In addition, enrichment for OCT and HOXC binding sites at the early time point and SORY matrices later after stimulation indicated an involvement of the phosphorylated members of the respective TF families in the execution of TLR-induced transcriptional responses. An initial test of the function for a few of these candidate transcriptional regulators was performed using siRNA knockdown in primary macrophages. These experiments suggested that knock down of the SORY binding phosphoprotein Capicua homolog (Cic) and to a lesser extent of the CREB family member Atf7 selectively attenuates LPS-induced expression of Il1a and Il1b.

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