Cross‐talk between phosphorylation and lysine acetylation in a genome‐reduced bacterium
2012; Springer Nature; Volume: 8; Issue: 1 Linguagem: Inglês
10.1038/msb.2012.4
ISSN1744-4292
AutoresVera van Noort, J. Seebacher, Samuel L. Bader, Shabaz Mohammed, Ivana Vonkova, Matthew J. Betts, Sebastian Kühner, Runjun D. Kumar, Tobias Maier, Martina O’Flaherty, Vladimir Rybin, Arne Schmeisky, Eva Yus, Jörg Stülke, Luís Serrano, Robert B. Russell, Albert J. R. Heck, Peer Bork, Anne‐Claude Gavin,
Tópico(s)Protein Structure and Dynamics
ResumoArticle28 February 2012Open Access Cross-talk between phosphorylation and lysine acetylation in a genome-reduced bacterium Vera van Noort Vera van Noort Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Jan Seebacher Jan Seebacher Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Samuel Bader Samuel Bader Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Shabaz Mohammed Shabaz Mohammed Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Ivana Vonkova Ivana Vonkova Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Matthew J Betts Matthew J Betts Cell Networks, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Sebastian Kühner Sebastian Kühner Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Runjun Kumar Runjun Kumar Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Tobias Maier Tobias Maier EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Martina O'Flaherty Martina O'Flaherty Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Vladimir Rybin Vladimir Rybin Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Arne Schmeisky Arne Schmeisky Department of General Microbiology, Georg-August University of Göttingen, Göttingen, Germany Search for more papers by this author Eva Yus Eva Yus EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Jörg Stülke Jörg Stülke Department of General Microbiology, Georg-August University of Göttingen, Göttingen, Germany Search for more papers by this author Luis Serrano Luis Serrano EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) and UPF, Barcelona, Spain ICREA, Pg. Lluís Companys 23, Barcelona, Spain Search for more papers by this author Robert B Russell Robert B Russell Cell Networks, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Albert JR Heck Albert JR Heck Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Peer Bork Corresponding Author Peer Bork Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Anne-Claude Gavin Corresponding Author Anne-Claude Gavin Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Vera van Noort Vera van Noort Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Jan Seebacher Jan Seebacher Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Samuel Bader Samuel Bader Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Shabaz Mohammed Shabaz Mohammed Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Ivana Vonkova Ivana Vonkova Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Matthew J Betts Matthew J Betts Cell Networks, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Sebastian Kühner Sebastian Kühner Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Runjun Kumar Runjun Kumar Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Tobias Maier Tobias Maier EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Martina O'Flaherty Martina O'Flaherty Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Vladimir Rybin Vladimir Rybin Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Arne Schmeisky Arne Schmeisky Department of General Microbiology, Georg-August University of Göttingen, Göttingen, Germany Search for more papers by this author Eva Yus Eva Yus EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) and UPF, Barcelona, Spain Search for more papers by this author Jörg Stülke Jörg Stülke Department of General Microbiology, Georg-August University of Göttingen, Göttingen, Germany Search for more papers by this author Luis Serrano Luis Serrano EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) and UPF, Barcelona, Spain ICREA, Pg. Lluís Companys 23, Barcelona, Spain Search for more papers by this author Robert B Russell Robert B Russell Cell Networks, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Albert JR Heck Albert JR Heck Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Peer Bork Corresponding Author Peer Bork Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Anne-Claude Gavin Corresponding Author Anne-Claude Gavin Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany Search for more papers by this author Author Information Vera van Noort1,‡, Jan Seebacher1,‡, Samuel Bader1,‡, Shabaz Mohammed2, Ivana Vonkova1, Matthew J Betts3, Sebastian Kühner1, Runjun Kumar1, Tobias Maier4, Martina O'Flaherty2, Vladimir Rybin1, Arne Schmeisky5, Eva Yus4, Jörg Stülke5, Luis Serrano4,6, Robert B Russell3, Albert JR Heck2, Peer Bork 1 and Anne-Claude Gavin 1 1Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Heidelberg, Germany 2Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands 3Cell Networks, University of Heidelberg, Heidelberg, Germany 4EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG) and UPF, Barcelona, Spain 5Department of General Microbiology, Georg-August University of Göttingen, Göttingen, Germany 6ICREA, Pg. Lluís Companys 23, Barcelona, Spain ‡These authors contributed equally to this work *Corresponding authors. Structural and Computational Biology Unit, European Molecular Biology Laboratory, EMBL, Meyerhofstrasse 1, Heidelberg 69117, Germany. Tel.: +49 6221 387 8816; Fax: +49 6221 387 517; E-mail: [email protected] or E-mail: [email protected] Molecular Systems Biology (2012)8:571https://doi.org/10.1038/msb.2012.4 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Protein post-translational modifications (PTMs) represent important regulatory states that when combined have been hypothesized to act as molecular codes and to generate a functional diversity beyond genome and transcriptome. We systematically investigate the interplay of protein phosphorylation with other post-transcriptional regulatory mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae. Systematic perturbations by deletion of its only two protein kinases and its unique protein phosphatase identified not only the protein-specific effect on the phosphorylation network, but also a modulation of proteome abundance and lysine acetylation patterns, mostly in the absence of transcriptional changes. Reciprocally, deletion of the two putative N-acetyltransferases affects protein phosphorylation, confirming cross-talk between the two PTMs. The measured M. pneumoniae phosphoproteome and lysine acetylome revealed that both PTMs are very common, that (as in Eukaryotes) they often co-occur within the same protein and that they are frequently observed at interaction interfaces and in multifunctional proteins. The results imply previously unreported hidden layers of post-transcriptional regulation intertwining phosphorylation with lysine acetylation and other mechanisms that define the functional state of a cell. Synopsis The effect of kinase, phosphatase and N-acetyltransferase deletions on proteome phosphorylation and acetylation was investigated in Mycoplasma pneumoniae. Bi-directional cross-talk between post-transcriptional modifications suggests an underlying regulatory molecular code in prokaryotes. Post-translational modifications (PTMs) change the chemical properties of proteins, conferring diversity beyond the amino-acid sequence. Proteins are often modified on multiple sites. A PTM code has been proposed, whereby modifications at specific positions influence further modifications. These regulatory circuits though have rarely been studied on a large-scale; conservation in prokaryotes remains elusive. Here, we studied two important PTMs– phosphorylation and lysine acetylation in the small bacterium Mycoplasma pneumoniae. We combined genetics and quantitative mass spectrometry to measure the effect of systematic kinase, phosphatase and N-acetyltransferase deletions on proteome abundance, phosphorylation and lysine acetylation. The data set represents a comprehensive analysis of both phosphorylation and lysine acetylation in a single prokaryote. It reveals (1) proteins often carry multiple modifications and multiple types of PTMs, reminiscent of the PTM code proposed in eukaryotes, (2) phosphorylation exerts pleiotropic effect on proteins abundances, phosphorylation, but also lysine acetylation, (3) the cross-talk between the two PTMs is bi-directional and (4) PTMs are frequently located at interaction interfaces and in multifunctional proteins, illustrating how PTMs could modulate protein functions affecting the way they interact. The study provides an unbiased and quantitative view on cross-talk between phosphorylation and lysine acetylation. It suggests that these regulatory circuits are a fundamental principle of regulation that might have evolved before the divergence of prokaryotes and eukaryotes. Introduction Cells constantly need to adapt their endogenous biochemical activities to a changing environment. An important level of adaptation is achieved by series of post-translational modifications (PTMs) that affect the chemical properties of proteins, conferring molecular diversity beyond the amino-acid sequence. More than 200 different PTMs have been described, and these are known to affect many aspects of protein function, such as activity, stability and interaction (Singh et al, 2007; Li et al, 2009; Deribe et al, 2010; Wang et al, 2010; Zhao et al, 2010). Among all PTMs, reversible protein phosphorylation and lysine acetylation represent prominent and ubiquitous regulatory mechanisms that are conserved from bacteria (Yu et al, 2008; Zhang et al, 2009a; Wang et al, 2010) to humans (Kim et al, 2006; Choudhary et al, 2009; Huttlin et al, 2010; Zhao et al, 2010). Protein phosphorylation is regulated by a variety of kinases and phosphatases, which are themselves regulated by phosphorylation within complex networks (Bodenmiller et al, 2010; Zorina et al, 2011). A series of mass spectrometry (MS)-based methods are currently available that allow the characterization of phosphorylation and lysine acetylation at unprecedented scales (Kim et al, 2006). Most recent analyses have captured 5000–10 000 phosphorylated (Van Hoof et al, 2009; Huttlin et al, 2010; Rigbolt et al, 2011) and 1700 lysine-acetylated (Choudhary et al, 2009) proteins, and large inventories of phosphorylation and acetylation sites are currently available (e.g., PHOSIDA (Gnad et al, 2011), Phospho.ELM (Dinkel et al, 2011) or PhosphoSite (Hornbeck et al, 2004)). In eukaryotes, many proteins have been observed to be modified on multiple sites, and some nuclear transcription factors (Yang and Seto, 2008b), cytoskeletal proteins (Reed et al, 2006; Zhang et al, 2009b) and protein chaperones (Scroggins et al, 2007) even carry several different PTMs, reminiscent of a molecular barcode. Inspired by histones, for which phosphorylation or lysine acetylation at specific positions influence further modifications and form complex regulatory circuits (Strahl and Allis, 2000; Jenuwein and Allis, 2001), the hypothesis of a protein modification code has been proposed, whereby dynamic patterns of protein modification would encode for alternative protein functions (Yang and Seto, 2008a). However, large-scale studies that consistently investigated direct modulation of one PTM by another are sparse (Yao et al, 2011). It is also unclear when such a code has evolved as prokaryotes are poorly studied in this respect with only a few instances of multiple PTMs in individual proteins having been previously identified (Soufi et al, 2008; Prisic et al, 2010). Accordingly, here we exhaustively studied two important PTM events—serine/threonine/tyrosine phosphorylation and lysine acetylation in the bacterium Mycoplasma pneumoniae, a human pathogen that causes atypical pneumonia (Waites and Talkington, 2004). This organism is established as a suitable model organism for large-scale systems-wide analyses on proteome, transcriptome, metabolic and protein networks (Guell et al, 2009; Kuhner et al, 2009; Yus et al, 2009; Maier et al, 2011). This self-replicating organism has one of the smallest known genomes (691 protein encoding genes) (Dandekar et al, 2000; Guell et al, 2009). It encodes a reduced PTM machinery, perturbation of which would reveal many of the regulatory cascades. Since it contains only one protein phosphatase (PrpC∣Mpn247), two known serine/threonine protein kinases (HprK∣Mpn223 and PknB∣PrkC∣Mpn248) and two putative N-acetyltransferases (Mpn027 and Mpn114), M. pneumoniae represents an ideal model organism in which to study system-wide impact of phosphorylation on other PTMs. We combined genetics and high-resolution quantitative MS to measure the global effect of kinase and phosphatase deletions on proteome abundance, phosphorylation and lysine acetylation. The study provides a first unbiased and quantifying view on cross-talk between phosphorylation and lysine acetylation and also suggests that these regulatory circuits are a fundamental principle of regulation that might have evolved before the divergence of prokaryotes and eukaryotes. Results Quantifying the M. pneumoniae proteome, phosphoproteome and lysine acetylome To gather insights into the mechanism of prokaryotic phosphorylation, and to systematically chart impacts of protein phosphorylation on lysine acetylation, we profiled both modifications in wild-type strains of M. pneumoniae and three isogenic mutants deficient in either one of the two protein kinases, HprK and PknB, or the phosphatase, PrpC (Halbedel et al, 2006) (Figure 1A). We applied a quantitative proteomics approach based on chemical, differential labeling with three isotopic dimethyl forms (Boersema et al, 2009). The chemically encoded digested proteomes (originating from the four strains) were combined according to a scheme that includes both technical and biological replicates to ensure that each proteome is chemically labeled with at least two different stable isotopes (Figure 1B; see Materials and methods). To reduce the complexity of the samples and increase sensitivity, peptides were subjected to fractionation: non-phosphorylated and phosphorylated peptides were separated by strong cation exchange (SCX) chromatography (Mohammed and Heck, 2010), whereas lysine-acetylated peptides were enriched using a specific antibody (Choudhary et al, 2009). All fractions were analyzed using a nano LC-LTQ-Orbitrap (Thermo, San Jose, CA) (see Materials and methods). Unmodified, phosphorylated and lysine-acetylated peptides were identified with the Mascot search engine using the M. pneumoniae sequence (UniProt) and corresponding decoy databases: peptide thresholds were set at false discovery rates (FDRs) of 1%. The majority (75%) of phosphorylation and all lysine acetylation sites could be localized to a single amino acid (see Materials and methods). Modified and unmodified peptides were quantified using the software MSQuant (Mortensen et al, 2010). Importantly, to prevent possible biases due to variation in protein expression, the relative intensities of modified peptides were normalized for changes in protein abundance (Figure 1C) (Wu et al, 2011). For each peptide, the statistical significance of the observed change in abundance was computed with the software OutlierD (Cho et al, 2008). The test provides a P-value based on the variation in the normalized ratios observed for all peptides with similar intensities (see Materials and methods). The thresholds were set stringently. Only changes that were statistically significant and that could be further confirmed by visual inspection were considered for further analysis. Additionally, changes in intensities lower than ∼2.8 × (log2<1.5, for proteins and phosphopeptides) or 4 × (log2 60% conserved in >80 eukaryotes) were frequently found in metabolic enzymes compared with other sites within other evolutionary ubiquitous proteins (P=2.0 × 10−4, Fisher's exact test). When considering exactly the same residues, lysine acetylation sites appear slightly less conserved than phosphorylation sites (P=2.8 × 10−3). However, when the acetylated-lysine was not conserved, an alternative lysine could be frequently found in other species within a window of three amino acids, one upstream or one downstream of the original aligned site (Figure 2D). This suggests that for some lysine acetylation sites, the exact position may not be so critical to maintain function. We also observed that proteins only occurring in species of the Mycoplasma genus were found to be less frequently modified than other proteins (Figure 2A), indicating that regulation through PTMs can evolve only secondary to proteome differentiation. The sites in these proteins likely represent recently acquired regulatory signals. Interestingly, none of these sites is conserved across all of the 12 sequenced Mycoplasma species, suggesting that they play species-specific regulatory functions. Dissecting the roles of kinases and phosphatase in M. pneumoniae phosphoproteome As consideration of putative changes in protein abundance is critical for the proper interpretation of PTM data (Wu et al, 2011), we first quantified the impact of kinase and phosphatase deletion on overall protein abundance. We observed that the levels of 39 of the 447 proteins quantified consistently were significantly affected (Supplementary Figure S4; Supplementary Table S1). We selected 15 for validation by western blot. Of the 45 abundances measured by western blotting in the three knockouts (k.o.), 39 (86.6%) showed upregulation and downregulation consistent with the previous MS data (Supplementary Figure S5). Among the proteins affected by PknB deletion, we found eight of the nine cytadherence proteins previously known to be downregulated upon PknB k.o. (Schmidl et al, 2010a) together with four uncharacterized proteins that could represent new players in the process of cell adhesion (Mpn256, Mpn387, Mpn400 and Mpn454; Supplementary Figure S4 and see Supplementary information). For proteins encoded by the cell-cycle operon: the ribosomal RNA small subunit methyltransferase H (MraW∣Mpn315), the cell-cycle proteins MraZ (Mpn314) and the tubulin-like protein FtsZ (Mpn317) we observed a decreased abundance upon PrpC deletion that correlated with changes in corresponding transcripts (Supplementary Table S4). However, in general, we found that mRNA levels were largely unaffected, indicating the existence of post-transcriptional regulatory mechanisms (Supplementary Table S4) (Schmidl et al, 2010a). These results show that perturbations of the phosphorylation network in M. pneumoniae affect protein abundance and turnover, acting at both transcriptional but also post-transcriptional levels. We also determined the impact of systematic kinase and phosphatase perturbation on the M. pneumoniae phosphoproteome (see Materials and methods). Of the 67 phosphosites unambiguously quantified, only 16 (23.9%) were never found to be affected (Supplementary Table S2). They might represent compensatory mechanisms, whereby deletion of one kinase may cause the other kinase to compensate. Alternatively, they might account for HprK-, PknB- and PrpC-independent phosphorylation events, including autophosphorylation of some metabolic enzymes (Jolly et al, 2000) or metabolic intermediates observed at catalytically active sites. For example, the constitutive phosphoserine (S64) in the ATP-binding site of the guanylate kinase (Gmk∣Mpn246) might represent a metabolic intermediate, since in the Escherichia coli structure the equivalent serine lies very close, though not obviously bound, to both phosphate and sulfate groups (Hible et al, 2005). However, the majority (76.1%) of the phosphosites were found regulated in at least one of the k.o., implying that the three enzymes are indeed the major modifiers of this network. The impact of protein kinase and phosphatase deletion largely depends on the extent of their substrate phosphorylation before perturbation,
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