Article pubs.acs.org/jpr
Global Phosphoproteomic Analysis Reveals Diverse Functions of Serine/Threonine/Tyrosine Phosphorylation in the Model Cyanobacterium Synechococcus sp. Strain PCC 7002 Ming-kun Yang,† Zhi-xian Qiao,†,‡ Wan-yi Zhang,† Qian Xiong,† Jia Zhang,† Tao Li,*,† Feng Ge,*,† and Jin-dong Zhao*,† †
Key Laboratory of Algal Biology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072, China University of Chinese Academy of Sciences, Beijing 100039, China
‡
S Supporting Information *
ABSTRACT: Increasing evidence shows that protein phosphorylation on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues is one of the major post-translational modifications in the bacteria, involved in regulating a myriad of physiological processes. Cyanobacteria are one of the largest groups of bacteria and are the only prokaryotes capable of oxygenic photosynthesis. Many cyanobacteria strains contain unusually high numbers of protein kinases and phosphatases with specificity on Ser, Thr, and Tyr residues. However, only a few dozen phosphorylation sites in cyanobacteria are known, presenting a major obstacle for further understanding the regulatory roles of reversible phosphorylation in this group of bacteria. In this study, we carried out a global and site-specific phosphoproteomic analysis on the model cyanobacterium Synechococcus sp. PCC 7002. In total, 280 phosphopeptides and 410 phosphorylation sites from 245 Synechococcus sp. PCC 7002 proteins were identified through the combined use of protein/ peptide prefractionation, TiO2 enrichment, and LC−MS/MS analysis. The identified phosphoproteins were functionally categorized into an interaction map and found to be involved in various biological processes such as two-component signaling pathway and photosynthesis. Our data provide the first global survey of phosphorylation in cyanobacteria by using a phosphoproteomic approach and suggest a wide-ranging regulatory scope of this modification. The provided data set may help reveal the physiological functions underlying Ser/Thr/Tyr phosphorylation and facilitate the elucidation of the entire signaling networks in cyanobacteria. KEYWORDS: Cyanobacteria, Synechococcus sp. PCC 7002, phosphoproteomics, two-component signaling pathway, photosynthesis
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INTRODUCTION Cyanobacteria, formerly referred to as blue-green algae, are the only prokaryotes capable of oxygenic photosynthesis using water as an electron donor.1 They are present in almost every habitat and play crucial roles in global carbon and nitrogen cycles.2 Cyanobacteria are ancient life forms and have successfully adapted to all habitats with a remarkable capacity to survive in a variety of extreme environments, such as high temperatures, extreme pH and high salinity.3 A representative of such organisms is the euryhaline, unicellular cyanobacterial strain Synechococcus sp. strain PCC 7002 (hereafter Synechococcus 7002). Synechococcus 7002 has become a model organism for studies of photosynthetic carbon fixation and biofuels development.4,5 It can tolerate extremely high-light intensities and grow over a wide range of NaCl concentrations.6 To cope with changing environmental conditions and survive in various harsh environments, Synechococcus 7002 has developed a complex signal transduction network to sense environmental signals and implement adaptive changes. © 2013 American Chemical Society
Reversible protein phosphorylation represents one of the most widespread post-translational modification (PTM) and a key mechanism by which environmental signals are transmitted to cause changes in protein expression or activity in both eukaryotic and prokaryotic cells.7 Genes encoding functional Ser/Thr/Tyr kinases are ubiquitous in prokaryotic genomes, and signaling through Ser/Thr/Tyr phosphorylation has emerged as an important regulatory mechanism in various microorganisms.8,9 Traditionally, the two-component system involving histidine kinases and their corresponding response regulators was considered as the predominant phosphorylationbased signal transduction mechanism in cyanobacteria.10 More recently, attention has focused on phosphorylation of proteins on Ser/Thr/Tyr residues in cyanobacteria. Many cyanobacterial strains contain unusually high numbers of protein kinases and phosphatases with specificity on Ser/Thr/Tyr residues, Received: January 3, 2013 Published: March 6, 2013 1909
dx.doi.org/10.1021/pr4000043 | J. Proteome Res. 2013, 12, 1909−1923
Journal of Proteome Research
Article
proteins involved in several important biological processes were functionally categorized into an interaction map. This is the first time that an interaction network of phosphoproteins in cyanobacteria was constructed, which may help us to better understand the significance of phosphorylation in key cellular mechanisms in cyanobacteria.
suggesting that these two phospho-based signaling systems are of comparable importance in this organism.11,12 For those fully sequenced cyanobacterial strains, the number of genes identified so far that encode Ser/Thr/Tyr kinases range from none to 52.11 For example, unicellular freshwater Synechocystis sp. PCC 6803 has 12 genes for putative Ser/Thr/Tyr kinases,13,14 whereas filamentous nitrogen-fixing Anabaena sp. PCC 7120 has 52 such genes.15,16 Inspection of the genome of Synechococcus 7002 revealed that it contains at least 12 Ser/ Thr/Tyr kinases and 6 Ser/Thr/Tyr protein phosphatases.4,5 Comprehensive functional analyses have revealed that Ser/ Thr/Tyr kinases and phosphatases are involved in the regulation of critical processes in cyanobacteria, such as stress adaptation, cell differentiation, cell motility, carbon and nitrogen metabolism and photosynthesis.11 Owing to the growing awareness of the importance of Ser/ Thr/Tyr phosphorylation in controlling various bacterial pathways, there have been attempts in the recent past decade to obtain the complete picture of the phosphoproteome of bacteria.17 Systemic studies of protein phosphorylation in bacteria commenced with the separation of phosphoproteins by 2D-gel electrophoresis.18 Starting from 2007, high accuracy mass spectrometry in combination with biochemical enrichment of phosphopeptides from digested cell lysates lead to the publication of the first site-specific Ser/Thr/Tyr phosphoproteome for the bacterium B. subtilis.19 The same approach has since been employed to analyze the model organisms E. coli20 and Lactococcus lactis21 and a number of bacterial pathogens,22−26 and has become the standard procedure in bacterial phosphoproteomics.27 Using this approach, we previously identified 163 phosphorylation sites on 84 proteins from the Gram-positive pathogenic bacterium Streptococcus pneumonia.24 These studies have generated large data sets of proteins phosphorylated on serine, threonine and tyrosine in bacteria, with identified phosphorylation sites which represent an excellent starting point for in-depth physiological characterization of kinases and their substrates. The first protein phosphorylation on Ser/Thr/Tyr residues in cyanobacteria was revealed by radioactive labeling of proteins in 1994.12 Since then, only a few cyanobacterial phosphoproteins have been identified to date and little progress has been made in ascertaining precisely which cyanobacterial proteins are phosphorylated, which kinases/phosphatases are involved, and what cellular processes are targeted by this post-translational modification.11,28 In order to fill this gap in our knowledge, we have initiated a systematic study of the identities and functional roles of the Ser/Thr/Tyr phosphoproteins in the Synechococcus 7002. The first step in the exploration of Ser/Thr/Tyr phosphoproteins in the member of cyanobacteria, which we present here, is a genome-wide and site-specific phosphoproteome analysis of Synechococcus 7002 using high accuracy mass spectrometry in combination with biochemical enrichment of phosphopeptides from digested cell lysates. The total outcome was the identification of 280 unique phosphopeptides from 245 Synechococcus 7002 proteins and the determination of 410 phosphorylation sites. This set of cyanobacterial proteins phosphorylated on Ser/Thr/Tyr residues is the first and largest available to date, supporting the emerging view that protein phosphorylation is a general and fundamental regulatory process, not restricted only to eukaryotes, and opens the way for its detailed functional and evolutionary analysis in cyanobacteria in general. Furthermore, the identified phospho-
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EXPERIMENTAL PROCEDURES
Cell Culture and Protein Extraction
The wild-type strain of Synechococcus 7002 was grown in medium A supplemented with 1 mg mL−1 NaNO3 as nitrogen source.4,29 The growth temperature was 38 °C with continuous illumination at 250 μmol photons m−2 s−1 and the liquid culture was bubbled with 1% (v/v) CO2 in air. Cells at the early/midexponential phase (OD730 nm ≈ 1.0−2.0) were harvested by centrifugation at 8,000 g for 15 min at 4 °C. The cells were washed twice with PBS buffer, resuspended in lysis buffer containing 20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 5 mM β-glycero-phosphate, 10 mM NaF, 1 mM Na3VO4, 10 mM Na4P2O7, 1× protease inhibitor cocktail and 1× phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA). The mixture was applied to sonication (2 s on, 2 s off) for about 30 min on ice with an output of 135 W (JY92-IIN, Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China). Cellular debris was removed by centrifugation at 12000× g for 30 min at 4 °C, and the resulting supernatants were stored in aliquots at −80 °C until further use. Protein concentrations were determined with Bradford assay. In-solution Trypsin Digestion and Reversed Phase C18 Column Prefractionation
Protein extracts (10 mg) were subjected to disulfide reduction with 25 mM of DTT (37 °C, 45 min) and alkylation with 50 mM of iodoacetamide (25 °C, 20 min in the dark). Proteins were precipitated with 5 volumes of ice-cold acetone, collected by centrifugation and washed with 80% ice-cold acetone. The pellet was redissolved in 50 mM of ammonium bicarbonate, digested with sequencing grade modified trypsin (1:100 w/w) (Promega, Madison, WI) at 37 °C for 4 h, and further digested with trypsin (1:100 w/w) at 37 °C for 20 h. The tryptic digestion was quenched by adding 0.1% trifluoroacetic acid (TFA) and dried in a vacuum centrifuge. Self-packed C18 columns were prepared by packing 500 mg of C18 material (40 μm, 60-Å pore size, Agilent Technologies) into 15 mL AGT Cleanert SPE columns (about 3 mL bed volume). The columns were washed five times with 10 mL of 100% acetonitrile (ACN), and then equilibrated with the same volume of 0.1% TFA. After the digested peptides were loaded sequentially onto three self-packed C18 columns, the columns were washed five times with 2.0 mL of 0.1% TFA for sample desalting and eluted with a series of elution buffers (2.0 mL) containing 0.1% TFA with different concentrations of ACN (10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60% and 100%). Fractions were collected and dried with a vacuum centrifuge and stored at −20 °C for further use. In-gel Trypsin Digestion
Protein extracts (15 mg) were resolved on a 8% SDS-PAGE gel (1.5 mm thick, 80 mm wide, 70 mm long), stained with Coomassie blue G250 and excised into 12 gel slices. Each slice was further diced into ∼1 mm3 cubes for in-gel digestion. Each section was washed in water and completely destained using 50 1910
dx.doi.org/10.1021/pr4000043 | J. Proteome Res. 2013, 12, 1909−1923
Journal of Proteome Research
Article
mM ammonium bicarbonate in 50% acetonitrile at room temperature. Proteins were disulfide reduced with 25 mM DTT for 30 min at 37 °C and alkylated for 10 min with 50 mM iodacetamide at room temperature in the dark. Digestion was carried out using 20 μg/mL sequencing grade modified trypsin in 50 mM NH4HCO3. Sufficient trypsin solution was added to swell the gel pieces, which were kept at 4 °C for 45 min and then incubated at 37 °C overnight. The supernatants were transferred into a new microcentrifuge tube and the gels were sonicated twice with extraction buffer (67% acetonitrile containing 5% trifluoroacetic acid). The peptide extract and the supernatant of the gel slice were combined and then completely dried in a vacuum centrifuge.
Both pFind and MASCOT were set up to search a database of forward and reversed Synechococcus 7002 protein database from Cyanobase (http://genome.kazusa.or.jp/cyanobase) containing 3186 protein sequences. The search criteria were as follows: trypsin digestion; carbamidomethylation (Cys) was set as fixed modification, whereas oxidation (M), phospho (ST), and phospho (Y) were considered as variable modifications; and two missed cleavages were allowed. Allowed maximum mass deviation was 0.4 Da (monoisotopic) for the precursor ion and fragment maximum mass deviation was 0.6 Da (monoisotopic). All phosphopeptide spectra identified by pFind were extracted with pBuild30−32 for further processing. The peptides shorter than seven amino acids were excluded. For search results, peptides with pFind E score 20 were accepted. The false discovery rate (FDR) of peptide identification was estimated of less than 1% according to the target-decoy strategy.33−35 All fragmentation spectra of putative phosphopeptides identified were manually verified using a method as described by Mann et al.19,20 The putative phosphopeptides were verified by the presence of at least three successive b- or y-ions. Simultaneously, the singly charged peptides containing Ser (P) and Thr (P) must at least have a lost phosphoric acid (−98 Da). In the phosphopeptides with multiple potential phosphorylation sites, the probabilities for phosphorylation at each potential site were calculated from the posttranslational modification (PTM) scores as described previously36,37 with slight modifications. In low energy CID, for the peptides containing RKNQ, peaks are seen for ions that have lost ammonia (−17 Da). For the peptides containing STED, the ions have lost water (−18 Da). The b and y ions including loss of ammonia and water were considered when we calculated the PTM score. Phosphorylation sites that were occupied with probability >0.75 were reported as class I phosphorylation sites and identified (“Localization p value” = 1 in Table S1, Supporting Information). Phosphorylation sites with localization probability
