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Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

ABSTRACT: Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in response to different stress situations. Quantitative analysis showed that a B. subtilis mutant lacking the YwlE arginine phosphatase accumulated a strikingly large number of arginine phosphorylations (217 sites in 134 proteins), however only a minor fraction of these sites was increasingly modified during heat shock or oxidative stress. The main targets of McsB-mediated arginine phosphorylation comprise central factors of the stress response system including the CtsR and HrcA heat shock repressors, as well as major components of the protein quality control system such as the ClpCP protease and the GroEL chaperonine. These findings highlight the impact of arginine phosphorylation in orchestrating the bacterial stress response.

SUBMITTER: Schmidt A

PROVIDER: S-EPMC3916652 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

Schmidt Andreas A Trentini Débora Broch DB Spiess Silvia S Fuhrmann Jakob J Ammerer Gustav G Mechtler Karl K Clausen Tim T

Molecular & cellular proteomics : MCP 20131120 2

Arginine phosphorylation is an emerging protein modification implicated in the general stress response of Gram-positive bacteria. The modification is mediated by the arginine kinase McsB, which phosphorylates and inactivates the heat shock repressor CtsR. In this study, we developed a mass spectrometric approach accounting for the peculiar chemical properties of phosphoarginine. The improved methodology was used to analyze the dynamic changes in the Bacillus subtilis arginine phosphoproteome in ...[more]

PMID: 24263382

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Project description:Application of a novel TiO2-based phosphopeptide enrichment protocol to study the role of protein arginine phosphorylation in B.subtilis. 1 iTRAQ Quantification of pArg peptides in B.subtilis upon heat shock and oxidative stress in comparison to unstressed control 2 Studying pArg upon deletion of McsB protein arginine kinase in B.subtilis (double mutant cells for McsB and YwlE phosphoarginine phosphatase) 3 Studying pArg in wt cells upon inhibition of YwlE phosphatase with cell wall permeable inihbitor sodium pervanadate Short overview McsB is a bacterial protein arginine kinase that is involve in stress adaptation for heat shock and oxidative stress - cells were grown in LB medium until O.D. 0.6 and split into 3 fractions - one fraction was treated with heat stress for 20 min at 50 degrees C, one was stressed with 1mM diamide (induced oxidative foldeing stress), and one was kept at 37 degrees C (control) - cell harvest, lysis with a modified FASP protocol including tryptic cleavage - iTRAQ labelling for some experiments - TiO2 enrichment to purify all phosphopeptides - LC-MS/MS analysis of phosphopetides - phosphosite localization by phosphoRS and manually - quantitation of phosphorylation relative to protein abundance in the different samples. Peptide and protein identification: Raw data were extracted by the Protein Discoverer software suite (version 1.2.227 for wt phosphorylation, all 4 replicates of the iTRAQ/TiO2 pArg study and replicates 1 and 2 for the pervanadate and the double mutant experiments and version 1.3.0.339 for protein abundance determination, and replicates 3 and 4 of the pervanadate and double mutant experiments. Thermo-Fisher Scientific) and searched against a combined forward/reversed database of B. subtilis protein sequences and common contaminants using MASCOT (version 2.2, www.matrixscience.com). Since cells were grown in LB medium, all raw data were independently searched against a combined B. subtilis and yeast (S. cerevisae) database which did not result in a significant increase in protein hits or identification of yeast proteins. Carbamidomethylation of cysteine, iTRAQ modification of peptide Nterminus and lysine;-amino group were set as fixed modifications. Phosphorylation of serine, threonine, tyrosine, histidine, and arginine as well as methionine sulfoxidation were selected to be variable, with a maximum of 4 modifications per peptide. Since tryptic cleavage is impaired at phosphorylated arginine, a maximum of two missed cleavage sites was allowed, whereas fully tryptic cleavage of both termini was required. The peptide mass deviation was set to 4 ppm; fragment ions were allowed to have a mass deviation of 0.5 Da for CID and ETD data and 0.025 Da for HCD data. False discovery rates were assessed from reversed database hits including all identified peptide spectrum matches with MASCOT score above 19, rank 1, and a peptide length of at least 6 amino acids. This resulted in false discovery rates below 1 % on the peptide spectrum match level and below 1.5% on the unique peptide level for each experiment. For reliable phosphorylation site analysis, all phosphopeptide hits were automatically re-analyzed by the phosphoRS software (Taus et al, 2011) within the Protein Discoverer software suite, manually validated, and compared to known B. subtilis protein phosphorylations (Macek et al, 2007). Proteins were grouped according to their biological function assignment on the SubtiList homepage (http://genolist.pasteur.fr/SubtiList/). Relative quantification of phosphorylation: Peptide quantification was achieved by the reporter ions quantifier node in Protein Discoverer. This node extracted reporter ion areas from corresponding HCD scans for all identified peptide hits (HCD, CID and ETD) based on the precursor mass. Extracted peak areas for heat shock and oxidative stress were divided by the peak area of the control sample to give the corresponding peptide abundance ratio. Regulatory cut-offs for peptide abundance ratios were determined from the distribution of unphosphorylated peptides. A 95% confidence window was applied to log2 normalized values of peptide regulations and all hits outside of this window were considered to be regulated. For the heat stress/control ratio regulatory cut-off values of 0.59 and 1.75 were determined. Peptides with abundance changes within 0.30 to 2.50 for the oxidative stress/control ratio were not considered to be regulated. Protein regulations were the averages of all identified and quantified unphosphorylated peptides from all analyzed samples (Supplementary Table 5). Phosphopeptide abundances were calculated by averaging, if a phosphopeptide was identified in several replicates and/or charge states. To argument for upregulation of phosphorylation, each phospho-peptide ratio was divided by the respective protein abundance ratio, in case the protein was identified before. For regulation of the phosphorylation, we oriented the regulatory cut-offs determined before. We compared each phosphopeptide abundance against the respective protein abundance and determined a twofold regulation to be significant.

Project description:Receptor interacting protein 3 (RIP3) is a protein kinase that plays a key role in programmed necrosis. Despite the importance of RIP3-dependent necrosis in many pathological processes, current knowledge on the function of RIP3 is very limited. Here we present the results of a proteome-wide analysis of RIP3-regulated phosphorylation sites using cells from wildtype (RIP3(+/+)) and RIP3 knockout (RIP3(-/-)) mice. Because the activation of RIP3 requires stimulation by certain extracellular stimuli such as ligands of death receptors or Toll-like receptors, we compared the phosphorylation sites of lipopolysaccharide (LPS)-treated peritoneal macrophages from RIP3(+/+) and RIP3(-/-) mice and the phosphorylation sites of tumor necrosis factor (TNF)-treated RIP3(+/+) and RIP3(-/-) mouse embryonic fibroblast (MEF) cells. Stable isotope labeling by amino acids in cell culture and spike-in stable isotope labeling by amino acids in cell culture were used in the analyses of the MEFs and macrophages, respectively. Proteomic analyses using stable isotope labeling by amino acids in cell culture coupled with immobilized metal affinity chromatography-hydrophilic interaction liquid chromatography fractionation and nanoLC MS/MS identified 14,057 phosphopeptides in 4306 proteins from the macrophages and 4732 phosphopeptides in 1785 proteins from the MEFs. Analysis of amino acid sequence motifs among the phosphopeptides identified a potential motif of RIP3 phosphorylation. Among the phosphopeptides identified, 73 were found exclusively in RIP3(+/+) macrophages, 121 were detected exclusively from RIP3(+/+) MEFs, 286 phosphopeptides were induced more in RIP3(+/+) macrophages than in RIP3(-/-) macrophages and 26 phosphopeptides had higher induction in RIP3(+/+) MEFs than in RIP3(-/-) cells. Many of the RIP3 regulated phosphoproteins from the macrophages and MEF cells are functionally associated with the cell cycle; the rest, however, appear to have diverse functions in that a number of metabolism related proteins were phosphorylated in macrophages and development related phosphoproteins were induced in MEFs. The results of our phosphoproteomic analysis suggest that RIP3 might function beyond necrosis and that cell type specific function of RIP3 exists.

Project description:Serine/threonine protein phosphatase 1 regulatory subunit 12A (PPP1R12A) modulates the activity and specificity of the catalytic subunit of protein phosphatase 1, regulating various cellular processes via dephosphorylation. Nonetheless, little is known about phosphorylation events controlled by PPP1R12A in skeletal muscle insulin signaling. Here, we used quantitative phosphoproteomics to generate a global picture of phosphorylation events regulated by PPP1R12A in a L6 skeletal muscle cell line, which were engineered for inducible PPP1R12A knockdown. Phosphoproteomics revealed 3876 phosphorylation sites (620 were novel) in these cells. Furthermore, PPP1R12A knockdown resulted in increased overall phosphorylation in L6 cells at the basal condition, and changed phosphorylation levels for 698 sites (assigned to 295 phosphoproteins) at the basal and/or insulin-stimulated conditions. Pathway analysis on the 295 phosphoproteins revealed multiple significantly enriched pathways related to insulin signaling, such as mTOR signaling and RhoA signaling. Moreover, phosphorylation levels for numerous regulatory sites in these pathways were significantly changed due to PPP1R12A knockdown. These results indicate that PPP1R12A indeed plays a role in skeletal muscle insulin signaling, providing novel insights into the biology of insulin action. This new information may facilitate the design of experiments to better understand mechanisms underlying skeletal muscle insulin resistance and type 2 diabetes.Biological significanceThese results identify a large number of potential new substrates of serine/threonine protein phosphatase 1 and suggest that serine/threonine protein phosphatase 1 regulatory subunit 12A indeed plays a regulatory role in multiple pathways related to insulin action, providing novel insights into the biology of skeletal muscle insulin signaling. This information may facilitate the design of experiments to better understand the molecular mechanism responsible for skeletal muscle insulin resistance and associated diseases, such as type 2 diabetes and cardiovascular diseases.

Project description:Heparan sulfate (HS) is a linear, abundant, highly sulfated polysaccharide that expresses in the vasculature. Recent genetic studies documented that HS critically modulates various endothelial cell functions. However, elucidation of the underlying molecular mechanism has been challenging because of the presence of a large number of HS-binding ligands found in the examined experimental conditions. In this report, we used quantitative phosphoproteomics to examine the global HS-dependent signaling by comparing wild type and HS-deficient endothelial cells that were cultured in a serum-containing medium. A total of 7222 phosphopeptides, corresponding to 1179 proteins, were identified. Functional correlation analysis identified 25 HS-dependent functional networks, and the top five are related to cell morphology, cellular assembly and organization, cellular function and maintenance, cell-to-cell communication, inflammatory response and disorder, cell growth and proliferation, cell movement, and cellular survival and death. This is consistent with cell function studies showing that HS deficiency altered endothelial cell growth and mobility. Mining for the underlying molecular mechanisms further revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, including ILK, integrin, actin cytoskeleton organization, tight junction and thrombin signaling. Intriguingly, this analysis unexpectedly determined that the top HS-dependent signaling is the IGF-1 signaling pathway, which has not been known to be modulated by HS. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/growth hormone signaling pathways, including those both previously known, such as HGF and VEGF, and those unknown, such as IGF-1, erythropoietin, angiopoietin/Tie, IL-17A and growth hormones. Twelve of the identified 22 growth factor/cytokine/growth hormone signaling pathways, including IGF-1 and angiopoietin/Tie signaling, were alternatively confirmed in phospho-receptor tyrosine kinase array analysis. In summary, our SILAC-based quantitative phosphoproteomic analysis confirmed previous findings and also uncovered novel HS-dependent functional networks and signaling, revealing a much broader regulatory role of HS on endothelial signaling.

Dataset Information

Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

Publications

Quantitative phosphoproteomics reveals the role of protein arginine phosphorylation in the bacterial stress response.

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