A proteomic approach to identify phosphoproteins encoded by cDNA libraries

We report a method for large-scale rapid analysis of phosphoproteins in tissues or cells by combining immobilized metal affinity chromatography (IMAC) with phage display cDNA library screening. We expressed a testis cDNA library as fusion proteins on phage and, using IMAC, enriched for sequences encoding phosphoproteins. Selected clones were polymerase chain reaction amplified and sequenced. The majority of the clones sequenced (80%) encoded known proteins previously identified as phosphoproteins. Immunoblotting with phosphotyrosine antibodies confirmed that some of the selected sequences encoded tyrosine phosphorylated proteins when expressed on phage. An advantage of this method is the rapid identification of phosphoproteins encoded by a cDNA library, which can identify proteins that are potentially phosphorylated in vivo. When this method is combined with limited enzymatic digestion and tandem mass spectrometric techniques, the specific phosphorylation site in a protein can be identified. This technique can be used in proteomics studies to effectively detect phosphorylated proteins and avoid time-consuming and expensive peptide sequencing.     

Qualitative and quantitative analyses of protein phosphorylation in naive and stimulated mouse synaptosomal preparations

Activity-dependent protein phosphorylation is a highly dynamic yet tightly regulated process essential for cellular signaling. Although recognized as critical for neuronal functions, the extent and stoichiometry of phosphorylation in brain cells remain undetermined. In this study, we resolved activity-dependent changes in phosphorylation stoichiometry at specific sites in distinct subcellular compartments of brain cells. Following highly sensitive phosphopeptide enrichment using immobilized metal affinity chromatography and mass spectrometry, we isolated and identified 974 unique phosphorylation sites on 499 proteins, many of which are novel. To further explore the significance of specific phosphorylation sites, we used isobaric peptide labels and determined the absolute quantity of both phosphorylated and non-phosphorylated peptides of candidate phosphoproteins and estimated phosphorylation stoichiometry. The analyses of phosphorylation dynamics using differentially stimulated synaptic terminal preparations revealed activity-dependent changes in phosphorylation stoichiometry of target proteins. Using this method, we were able to differentiate between distinct isoforms of Ca2+/calmodulin-dependent protein kinase (CaMKII) and identify a novel activity-regulated phosphorylation site on the glutamate receptor subunit GluR1. Together these data illustrate that mass spectrometry-based methods can be used to determine activity-dependent changes in phosphorylation stoichiometry on candidate phosphopeptides following large scale phosphoproteome analysis of brain tissue.     

Identification of phosphoproteins and their phosphorylation sites in the WEHI-231 B lymphoma cell line

A major goal of the Alliance for Cellular Signaling is to elaborate the components of signal transduction networks in model cell systems, including murine B lymphocytes. Due to the importance of protein phosphorylation in many aspects of cell signaling, the initial efforts have focused on the identification of phosphorylated proteins. In order to identify serine- and threonine-phosphorylated proteins on a proteome-wide basis, WEHI-231 cells were treated with calyculin A, a serine/threonine phosphatase inhibitor, to induce high levels of protein phosphorylation. Proteins were extracted from whole-cell lysates and digested with trypsin. Phosphorylated peptides were then enriched using immobilized metal affinity chromatography and identified by liquid chromatography-tandem mass spectrometry. A total of 107 proteins and 193 phosphorylation sites were identified using these methods. Forty-two of these proteins have been reported to be phosphorylated, but only some of them have been detected in B cells. Fifty-four of the identified proteins were not previously known to be phosphorylated. The remaining 11 phosphoproteins have previously only been characterized as novel cDNA or genomic sequences. Many of the identified proteins were phosphorylated at multiple sites. The proteins identified in this study significantly expand the repertoire of proteins known to be phosphorylated in B cells. The number of newly identified phosphoproteins indicates that B cell signaling pathways utilizing protein phosphorylation are likely to be more complex than previously appreciated.     

Possible involvements of 101 kDa, 90 kDa, and 32 kDa phosphoproteins in the phase-shift of Dictyostelium cells from growth to differentiation

Abstract. As demonstrated previously, the transition of starving Dictyostelium cells from growth to differentiation phase occurs at a particular position (putative shift point; PS-point) in G₂-phase of the cell cycle of Dictyostelium discoideum Ax-2. In this study we examined what proteins are phosphorylated or dephosphorylated at the onset of starvation, with special emphasis on changes of phosphoproteins near the PS-point. When AX-2 cells at any particular phase of the cell cycle were pulse-labeled with inorganic ³²P (³²P_i) in the presence or absence of nutrients, it was found that 101 kDa and 90 kDa phosphoproteins exhibit specific changes around the PS-point. From the chase-experiments of ³²P-labeled cells, the 101 kDa and 90 kDa proteins were found to fail to be phosphorylated at the PS-point under starvation conditions. The protein phosphatase inhibitors such as okadaic acid and calyculin A inhibited completely entry of starving Ax-2 cells to differentiation, and also blocked perfectly dephosphorylation of 32 kDa protein. Taken together it is likely that dephosphorylation of 32 kDa protein as well as low phosphorylation levels of 101 kDa and 90 kDa proteins may be required for the phase-shift of Ax-2 cells from growth to differentiation. Subcellular fractionation showed the 101 kDa phosphoprotein to be located in cytoplasm, while parts, at least, of the 90 kDa and 32 kDa phosproproteins were in the nucleus. In addition, the results of cellulose thin-layer electrophoresis of digested 101 kDa and 90 kDa phosphoproteins show that in both proteins only serine residues are phosphorylated. The significance of phosphorylation states of 101 kDa, 90 kDa, and 32 kDa proteins is discussed in relation to a breakaway of cells from proliferation to differentiation.     

Conserved phosphorylation of serines in the Ser-X-Glu/Ser(P) sequences of the vitamin K-dependent matrix Gla protein from shark, lamb, rat, cow, and human.

The present studies demonstrate that matrix Gla protein (MGP), a 10-kDa vitamin K-dependent protein, is phosphorylated at 3 serine residues near its N-terminus. Phosphoserine was identified at residues 3, 6, and 9 of bovine, human, rat, and lamb MGP by N-terminal protein sequencing. All 3 modified serines are in tandemly repeated Ser-X-Glu sequences. Two of the serines phosphorylated in shark MGP, residues 2 and 5, also have glutamate residues in the n + 2 position in tandemly repeated Ser-X-Glu sequences, whereas the third, shark residue 3, would acquire an acidic phosphoserine in the n + 2 position upon phosphorylation of serine 5. The recognition motif found for MGP phosphorylation, Ser-X-Glu/Ser(P), has been seen previously in milk caseins, salivary proteins, and a number of regulatory peptides. A review of the literature has revealed an intriguing dichotomy in the extent of serine phosphorylation among secreted proteins that are phosphorylated at Ser-X-Glu/Ser(P) sequences. Those phosphoproteins secreted into milk or saliva are fully phosphorylated at each target serine, whereas phosphoproteins secreted into the extracellular environment of cells are partially phosphorylated at target serine residues, as we show here for MGP and others have shown for regulatory peptides and the insulin-like growth factor binding protein 1. We propose that the extent of serine phosphorylation regulates the activity of proteins secreted into the extracellular environment of cells, and that partial phosphorylation can therefore be explained by the need to ensure that the phosphoprotein be poised to gain or lose activity with regulated changes in phosphorylation status.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphoproteome analysis of the human Chang liver cells using SCX and a complementary mass spectrometric strategy

The liver is the largest organ in the body, with many complex, essential functions, such as metabolism, deintoxication, and secretion, often regulated via post-translational modifications, especially phosphorylation. Thus, the detection of phosphoproteins and phosphorylation sites is important to comprehensively explore human liver biological function. The human Chang liver cell line is among the first derived from non-malignant tissue, and its phosphoproteome profile has never been globally analyzed. To develop the complete phosphoproteome and probe the roles of protein phosphorylation in normal human liver, we adopted a shotgun strategy based on strong cation exchange chromatograph, titanium dioxide and LC-MS/MS to isolate and identify phosphorylated proteins. Two types of MS approach, Q-TOF and IT, were used and compared to identify phosphosites from complex protein mixtures of these cells. A total of 1035 phosphorylation sites and 686 phosphorylated peptides were identified from 607 phosphoproteins. A search using the public database of PhosphoSite showed that approximately 344 phosphoproteins and 760 phosphorylation sites appeared to be novel. In addition, N-terminal phosphorylated peptides were a greater fraction of all identified phosphopeptides. With GOfact analysis, we found that most of the identified phosphoproteins are involved in regulating metabolism, consistent with the liver's role as a key metabolic organ.     

Exosome‐mediated extracellular release of polyadenylate‐binding protein 1 in human metastatic duodenal cancer cells

Exosomes are small vesicles secreted from cells that transport their embedded molecules through bidirectional exocytosis‐ and endocytosis‐like pathways. Expression patterns of exosomal molecules such as proteins and RNAs can be indicative of cell type since their signature is thought to be unique among cells. Using human primary (AZ‐521) and metastatic (AZ‐P7a) duodenal cancer cell lines, we conducted a comparative exosomal proteome analysis to identify proteins with metastatic marker potential. As determined by LC‐MS/MS and Western blot analyses, polyadenylate‐binding protein 1 (PABP1) was found to be predominantly abundant in AZ‐P7a exosomes. The amount of exosomal PABP1 in AZ‐P7a cells increased by treating the cells with inhibitors for the classical ER/Golgi secretory pathway (brefeldin A and monensin) and the ubiquitin‐proteasome pathway (MG‐132 and PYR‐41). Treatment of AZ‐P7a cells with the neutral sphingomyelinase inhibitor GW4869, which suppresses exosome release, not only reduced the amount of exosomal PABP1 but also produced PABP1‐immunoreactive products cleaved via a proteolysis‐like process. Taken together, these results suggest that AZ‐P7a cells do not tolerate intracellular PABP1 accumulation and are thus exported into the extracellular milieu by the exosome‐mediated pathway. In addition, PABP1 has a potential use as a biomarker for metastatic duodenal cancer.     

Diverse mitogenic agents induce the phosphorylation of two related 42,000-dalton proteins on tyrosine in quiescent chick cells.

Incubation of quiescent chicken embryo cells with platelet-derived growth factor, epidermal growth factor, or serum was found to stimulate phosphorylation of two proteins of ca. 42,000 daltons on tyrosine. These proteins are structurally related to each other and to two proteins phosphorylated on tyrosine under similar conditions in mitogen-treated mouse fibroblasts. Three other very different mitogenic agents, the protease trypsin and the chemically unrelated tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and teleocidin, stimulated phosphorylation of the same proteins. In all cases, phosphotyrosine was detected in these phosphoproteins. Although additional changes in protein phosphorylation were evident, no other proteins were observed by two-dimensional gel electrophoresis which contained increased amounts of phosphotyrosine in mitogen-treated chicken embryo cells. One of these 42,000-dalton proteins was shown previously to be phosphorylated on tyrosine in chicken embryo cells transformed with various retroviruses whose transforming proteins possess tyrosine protein kinase activity. Phosphorylation of the 42,000-dalton proteins could be important in the regulation of cell division.     

Analysis of the Arabidopsis cell suspension phosphoproteome in response to short-term low temperature and abscisic acid treatment

To shed light on the early protein phosphorylation events involved in plant cell signaling in response to environmental stresses, we studied changes in the phosphorylation status of the Arabidopsis cell suspension proteome after short-term low temperature and abscisic acid (ABA) treatment. We used radioactive pulse-labeling of Arabidopsis cell suspension cultures and two-dimensional (2-D) gel electrophoresis to identify proteins that are differentially phosphorylated in response to these treatments. Changes in the phosphorylation levels of several proteins were detected in response to short-term (5 min or less) cold (4°C) and chilling (12°C) stress and ABA treatment, and we observed that some of these changes were common between these treatments. In addition, we used Pro-Q Diamond phosphoprotein gel stain to study the steady-state protein phosphorylation status under the same treatments. We demonstrated that Pro-Q Diamond effectively stained phosphorylated proteins, however, the overall Pro-Q Diamond 2-D gel staining pattern of proteins extracted from low-temperature and ABA-treated cells was not consistent with the gel patterns obtained by in vivo radioactive labeling of phosphoproteins. These in vivo pulsed-labeling experiments demonstrate that the Arabidopsis phosphoproteome is dynamic in response to short-term low temperature and ABA treatment, and thus represents a strategy for the identification of signaling proteins that could be utilized in the production of chilling or freeze tolerant crop varieties.     

Comprehensive phosphoproteome analysis in rice and identification of phosphoproteins responsive to different hormones/stresses

Phosphoproteins in rice were detected by in vitro protein phosphorylation followed by two-dimensional polyacrylamide gel electrophoresis. Forty-four phosphoproteins were detected on a 2D-gel after in vitro protein phosphorylation of the crude extract from rice leaf sheath. Among the phosphoproteins detected, 42 were identified through analysis by Q-TOF MS/MS and/or MALDI-TOF MS. The largest percentage of the identified phosphoproteins are involved in signaling (30%), while 18% are involved in metabolism. When rice seedlings were treated with various hormones and stresses, it was observed that the phosphorylation of 13 proteins was enhanced differentially by different hormone and stress treatments. Furthermore, when the hormone/stress regulated phosphoproteins are compared in rice leaf sheath, leaf blade and root, only cytoplasmic malate dehydrogenase was found to be phosphorylated in all the tissues. Results suggest that in the phosphorylation cascade of rice, glycolytic metabolism processes and Ca(2+)-signaling seem to be important targets in response to hormones and stresses. Furthermore, the direct visualization of phosphoproteins by (32)P-labeling and their mass spectrometric identification provides an accurate and reliable method of analyzing the rice phosphoproteome.     

Enrichment and Analysis of Intact Phosphoproteins in Arabidopsis Seedlings

Protein phosphorylation regulates diverse cellular functions and plays a key role in the early development of plants. To complement and expand upon previous investigations of protein phosphorylation in Arabidopsis seedlings we used an alternative approach that combines protein extraction under non-denaturing conditions with immobilized metal-ion affinity chromatography (IMAC) enrichment of intact phosphoproteins in Rubisco-depleted extracts, followed by identification using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). In-gel trypsin digestion and analysis of selected gel spots identified 144 phosphorylated peptides and residues, of which only18 phosphopeptides and 8 phosphosites were found in the PhosPhAt 4.0 and P³DB Arabidopsis thaliana phosphorylation site databases. More than half of the 82 identified phosphoproteins were involved in carbohydrate metabolism, photosynthesis/respiration or oxidative stress response mechanisms. Enrichment of intact phosphoproteins prior to 2-DE and LC-MS/MS appears to enhance detection of phosphorylated threonine and tyrosine residues compared with methods that utilize peptide-level enrichment, suggesting that the two approaches are somewhat complementary in terms of phosphorylation site coverage. Comparing results for young seedlings with those obtained previously for mature Arabidopsis leaves identified five proteins that are differentially phosphorylated in these tissues, demonstrating the potential of this technique for investigating the dynamics of protein phosphorylation during plant development.     

The stoichiometry of protein phosphorylation in adipocyte lipid droplets: Analysis by N‐terminal isotope tagging and enzymatic dephosphorylation

Most phosphoproteomic studies to date have been limited to the identification of phosphoproteins and their phosphorylation sites, and have not assessed the stoichiometry of protein phosphorylation, a critical parameter reflecting the dynamic equilibrium between phosphorylated and non‐phosphorylated pools of proteins. Here, we used a method for measuring phosphorylation stoichiometry through isotope tagging and enzymatic dephosphorylation of tryptic peptides. Using this method, protein digests are divided into two equal aliquots that are modified with either light or heavy isotope tags. One aliquot is dephosphorylated by alkaline phosphatase. Finally, the peptide mixtures are recombined and LC‐MS/MS analysis is performed. With this method, we studied adipocytes of mice stimulated with CL316,243, a β‐3 adrenergic agonist known to induce lipolysis and marked phosphorylation changes in proteins of the lipid droplet surface. In lipid droplet preparations, CL316,243 administration increased phosphorylation of proteins related to regulation of signaling, metabolism and intracellular trafficking in white adipose tissue, including hormone‐sensitive lipase which was 80% phosphorylated at the previously reported site, Ser‐559, and the lipid surface protein perilipin, which was phosphorylated by ～60 and ～40% at previously unreported sites, Ser‐410 and Ser‐460.     

A phosphoproteomic landscape of rice (Oryza sativa) tissues

Protein phosphorylation is an important posttranslational modification that regulates various plant developmental processes. Here, we report a comprehensive, quantitative phosphoproteomic profile of six rice tissues, including callus, leaf, root, shoot meristem, young panicle and mature panicle from Nipponbare by employing a mass spectrometry (MS)‐based, label‐free approach. A total of 7171 unique phosphorylation sites in 4792 phosphopeptides from 2657 phosphoproteins were identified, of which 4613 peptides were differentially phosphorylated (DP) among the tissues. Motif‐X analysis revealed eight significantly enriched motifs, such as [sP], [Rxxs] and [tP] from the rice phosphosites. Hierarchical clustering analysis divided the DP peptides into 63 subgroups, which showed divergent spatial‐phosphorylation patterns among tissues. These clustered proteins are functionally related to nutrition uptake in roots, photosynthesis in leaves and tissue differentiation in panicles. Phosphorylations were specific in the tissues where the target proteins execute their functions, suggesting that phosphorylation might be a key mechanism to regulate the protein activity in different tissues. This study greatly expands the rice phosphoproteomic dataset, and also offers insight into the regulatory roles of phosphorylation in tissue development and functions.     

Skeletal muscle sarcolemma proteins as targets for adenosine 3':5'-monophosphate-dependent and calcium-dependent protein kinases

The present study documents the existence in rat skeletal muscle plasma membrane (sarcolemma) of a distinct set of proteins, most of which represent unknown protein species, which can be phosphorylated in vitro by addition of cAMP-dependent or calcium-dependent protein kinases. Under the experimental conditions used, cAMP-regulated protein phosphorylation appeared to be the most important phosphorylation system in these membranes, followed by the calcium/phospholipid-regulated, and, with only a few substrates detected, the calcium/calmodulin-regulated systems. No specific substrate for cGMP-dependent protein kinase was found. In contrast, calcium/calmodulin-regulated protein phosphorylation was the most important in the sarcoplasmic reticulum fraction. Most of the cAMP-regulated and calcium/phospholipid-regulated sarcolemma phosphoproteins appeared to be intrinsic membrane proteins, at least three of which appeared to be phosphorylated by both these protein kinases. These phosphoproteins may represent membrane targets for multiple hormone or transmitter actions in skeletal muscle cells. Our results, therefore, suggest that protein phosphorylation systems, particularly those regulated by cAMP or calcium/phospholipid, may be more important in the regulation of sarcolemma function than hitherto believed.     

Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines

Exosomes are important mediators in cell‐to‐cell communication and, recently, their role in melanoma progression has been brought to light. Here, we characterized exosomes secreted by seven melanoma cell lines with varying degrees of aggressivity. Extensive proteomic analysis of their exosomes confirmed the presence of characteristic exosomal markers as well as melanoma‐specific antigens and oncogenic proteins. Importantly, the protein composition differed among exosomes from different lines. Exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response, while these proteins were less abundant or absent in exosomes from less aggressive cells. Interestingly, when exposed to exosomes from metastatic lines, less aggressive cells increased their migratory capacities, likely due to transfer of pro‐migratory exosomal proteins to recipient cells. Hence, this study shows that the specific protein composition of melanoma exosomes depends on the cells’ aggressivity and suggests that exosomes influence the behavior of other tumor cells and their microenvironment.     

Protein kinases phosphorylate long disordered regions in intrinsically disordered proteins

Phosphorylation is a major post‐translational modification that plays a central role in signaling pathways. Protein kinases phosphorylate substrates (phosphoproteins) by adding phosphate at Ser/Thr or Tyr residues (phosphosites). A large amount of data identifying and describing phosphosites in phosphoproteins has been reported but the specificity of phosphorylation is not fully resolved. In this report, data of kinase‐substrate pairs identified by the Kinase‐Interacting Substrate Screening (KISS) method were used to analyze phosphosites in intrinsically disordered regions (IDRs) of intrinsically disordered proteins. We compared phosphorylated and nonphosphorylated IDRs and found that the phosphorylated IDRs were significantly longer than nonphosphorylated IDRs. The phosphorylated IDR is often the longest IDR (71%) in a phosphoprotein when only a single phosphosite exists in the IDR, and when the phosphoprotein has multiple phosphosites in an IDR(s), the phosphosites are primarily localized in a single IDR (78%) and this IDR is usually the longest one (81%). We constructed a stochastic model of phosphorylation to estimate the effect of IDR length. The model that accounted for IDR length produced more realistic results when compared with a model that excluded the IDR length. We propose that the IDR length is a significant determinant for locating kinase phosphorylation sites in phosphoproteins.     

Identification of extracellularly phosphorylated membrane proteins

Ecto‐protein kinases phosphorylate extracellular membrane proteins and exhibit similarities to casein kinases and protein kinases A and C. However, the identification of their protein substrates still remains a challenge because a clear separation from intracellular phosphoproteins is difficult. Here, we describe a straightforward method for the identification of extracellularly phosphorylated membrane proteins in human umbilical vein endothelial cells (HUVECs) and K562 cells which used the protease bromelain to selectively remove ectoproteins from intact cells and combined this with the subsequent analysis using IMAC and LC‐MS/MS. A “false‐positive” strategy in which cells without protease treatment served as controls was applied. Using this approach we identified novel phosphorylation sites on five ectophosphoproteins (NOTCH1, otopetrin 1, regulator of G‐protein signalling 13 (RGS13), protein tyrosine phosphatase receptor type D isoform 3 (PTPRD), usherin isoform B (USH2A)). Use of bromelain appears to be a reliable technique for the further identification of phosphorylated surface‐exposed peptides when extracellular adenosine‐5'‐triphosphate is elevated during purinergic signalling.     

Calnexin phosphorylation: Linking cytoplasmic signalling to endoplasmic reticulum lumenal functions

Calnexin is an abundant integral membrane phosphoprotein of the endoplasmic reticulum (ER) of eukaryotic cells. The role of the luminal domain as an N-glycoprotein specific lectin has been well-established. Cytosolic C-terminal domain phosphorylation of calnexin has recently been elucidated in glycoprotein folding and quality control. Signalling of the presence of unfolded proteins from the lumen of the ER is mediated by the three ER membrane sensor proteins Ire1, ATF6 and PERK. The observation that the C-terminus of calnexin is differentially phosphorylated when glycoproteins are misfolded initiated our search for functional roles of calnexin phosphorylation. Recent studies have defined a role for phosphorylation at a proline-directed kinase site (Ser563) in ER protein quality control, while phosphorylation at a casein kinase 2 site (Ser534, Ser544) may be linked to transport functions. There are also four other abundant integral membrane phosphoproteins in the ER, and these may be components of other signalling pathways that link and coordinate other ER functions with the rest of the cell.     

Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation

Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is generally considered the major regulatory posttranslational modification in eukaryotic cells. Increasing evidence at the genome and proteome level shows that this modification is also present and functional in prokaryotes. We have recently reported the first in-depth phosphorylation site-resolved dataset from the model Gram-positive bacterium, Bacillus subtilis, showing that Ser/Thr/Tyr phosphorylation is also present on many essential bacterial proteins. To test whether this modification is common in Eubacteria, here we use a recently developed proteomics approach based on phosphopeptide enrichment and high accuracy MS to analyze the phosphoproteome of the model Gram-negative bacterium Escherichia coli. We report 81 phosphorylation sites on 79 E. coli proteins, with distribution of Ser/Thr/Tyr phosphorylation sites 68%/23%/9%. Despite their phylogenetic distance, phosphoproteomes of E. coli and B. subtilis show striking similarity in size, classes of phosphorylated proteins, and distribution of Ser/Thr/Tyr phosphorylation sites. By combining the two datasets, we created the largest phosphorylation site-resolved database of bacterial phosphoproteins to date (available at www.phosida.com) and used it to study evolutionary conservation of bacterial phosphoproteins and phosphorylation sites across the phylogenetic tree. We demonstrate that bacterial phosphoproteins and phosphorylated residues are significantly more conserved than their nonphosphorylated counterparts, with a number of potential phosphorylation sites conserved from Archaea to humans. Our results establish Ser/Thr/Tyr phosphorylation as a common posttranslational modification in Eubacteria, present since the onset of cellular life.