Bacterial phosphoproteomic analysis reveals the correlation between protein phosphorylation and bacterial pathogenicity

Increasing evidence shows that protein phosphorylation on serine, threonine and tyrosine residues is a major regulatory post-translational modification in the bacteria. This review focuses on the implications of bacterial phosphoproteome in bacterial pathogenicity and highlights recent development of methods in phosphoproteomics and the connectivity of the phosphorylation networks. Recent technical developments in the high accuracy mass spectrometry have dramatically transformed proteomics and made it possible the characterization of a few exhaustive site-specific bacterial phosphoproteomes. The high abundance of tyrosine phosphorylations in a few bacterial phosphoproteomes suggests their roles in the pathogenicity, especially in the case of pathogen-host interactions; the high abundance of multi-phosphorylation sites in bacterial phosphoprotein is a compensation of the relatively small phosphorylation size and an indicator of the delicate regulation of protein functions.     

Impact of phosphoproteomics on studies of bacterial physiology

Protein phosphorylation on serine, threonine and tyrosine is recognized as a major tool of signal transduction in bacteria. However, progress in the field has been hampered by the lack of global and site-specific data on bacterial phosphoproteomes. Recent advances in mass spectrometry-based proteomics have encouraged bacteriologists to start using powerful gel-free approaches for global detection of phosphorylated proteins. These studies have generated large data sets of proteins phosphorylated on serine, threonine and tyrosine, with identified phosphorylation sites which represent an excellent starting point for in-depth physiological characterization of kinases and their substrates. The list of phosphorylated proteins inspired a number of physiological studies in which the identity of the phosphorylation site facilitated the elucidation of molecular mechanisms of signaling and regulation. Bacterial phosphoproteomics also provided interesting insights into the evolutionary aspects of protein phosphorylation. The field is rapidly embracing quantitative mass spectrometry strategies, comparing phosphoproteome dynamics in changing conditions and aiming to reconstruct the entire regulatory networks by linking kinases to their physiological substrates.     

Functional Divergence and Evolutionary Turnover in Mammalian Phosphoproteomes

Protein phosphorylation is a key mechanism to regulate protein functions. However, the contribution of this protein modification to species divergence is still largely unknown. Here, we studied the evolution of mammalian phosphoregulation by comparing the human and mouse phosphoproteomes. We found that 84% of the positions that are phosphorylated in one species or the other are conserved at the residue level. Twenty percent of these conserved sites are phosphorylated in both species. This proportion is 2.5 times more than expected by chance alone, suggesting that purifying selection is preserving phosphoregulation. However, we show that the majority of the sites that are conserved at the residue level are differentially phosphorylated between species. These sites likely result from false-negative identifications due to incomplete experimental coverage, false-positive identifications and non-functional sites. In addition, our results suggest that at least 5% of them are likely to be true differentially phosphorylated sites and may thus contribute to the divergence in phosphorylation networks between mouse and humans and this, despite residue conservation between orthologous proteins. We also showed that evolutionary turnover of phosphosites at adjacent positions (in a distance range of up to 40 amino acids) in human or mouse leads to an over estimation of the divergence in phosphoregulation between these two species. These sites tend to be phosphorylated by the same kinases, supporting the hypothesis that they are functionally redundant. Our results support the hypothesis that the evolutionary turnover of phosphorylation sites contributes to the divergence in phosphorylation profiles while preserving phosphoregulation. Overall, our study provides advanced analyses of mammalian phosphoproteomes and a framework for the study of their contribution to phenotypic evolution.     

PhosphoBlast, a computational tool for comparing phosphoprotein signatures among large datasets

Identification of specific protein phosphorylation sites provides predicative signatures of cellular activity and specific disease states such as cancer, diabetes, Alzheimer disease, and rheumatoid arthritis. Recent progress in phosphopeptide isolation technology and tandem mass spectrometry has provided the means to identify thousands of phosphorylation sites from a single biological sample. These advances now make it possible to profile global changes in the phosphoproteome at an unprecedented level. However, although this technology is generating a wealth of information, there is currently no efficient means to identify phosphoprotein signatures shared among large phosphoprotein databases. Identification of common phosphoprotein signatures found in biologically relevant systems and their conservation throughout evolution would provide valuable insight into mechanisms of signal transduction and cell function. Here we describe the development of a computational program (PhosphoBlast) that can rapidly match thousands of phosphopeptides that share phosphorylation sites within and across species. PhosphoBlast analysis of several large phosphoprotein datasets from the literature revealed common phosphorylation signatures shared across diverse experimental platforms and species. Moreover PhosphoBlast is a powerful analysis tool to identify specific phosphosite mutations. Comparison of the mouse and human phosphoproteomes revealed more than 130 specific phosphoamino acid mutations, some of which are predicted to alter protein function. Further analysis revealed that known phosphorylated amino acids are more evolutionally conserved than the Ser/Thr/Tyr amino acids not known to be phosphorylated. Together our results demonstrate that PhosphoBlast is a versatile mining tool capable of identifying related phosphorylation signatures and phosphoamino acid mutations among complex proteomics datasets in a highly efficient and accurate manner. PhosphoBlast will aid in the informatics analysis of the phosphoproteome and the identification of phosphoprotein biomarkers of disease.     

Site-specific analysis of bacterial phosphoproteomes

Protein phosphorylation on serine, threonine and tyrosine is established as an important regulatory modification in bacteria. A growing number of studies employing mass spectrometry-based proteomics report large protein phosphorylation datasets, providing precise evidence for in-vivo phosphorylation that is especially suitable for functional follow-up. Here, we provide an overview of the strategies currently used in bacterial phosphoproteomics, with an emphasis on gel-free proteomics and approaches that enable global detection of phosphorylation sites in bacterial proteins. The proteomics technology has matured sufficiently to permit routine characterization of phosphoproteomes and phosphopeptides with high sensitivity; we argue that the next challenge in the field will be the large-scale detection of protein kinase and phosphatase substrates and their integration into regulatory networks of the bacterial cell.     

Charging it up: global analysis of protein phosphorylation

Protein phosphorylation affects most, if not all, cellular activities in eukaryotes and is essential for cell proliferation and development. An estimated 30% of cellular proteins are phosphorylated, representing the phosphoproteome, and phosphorylation can alter a protein's function, activity, localization and stability. Recent studies for large-scale identification of phosphosites using mass spectrometry are revealing the components of the phosphoproteome. The development of new tools, such as kinase assays using modified kinases or protein microarrays, enables rapid kinase substrate identification. The dynamics of specific phosphorylation events can now be monitored using mass spectrometry, single-cell analysis of flow cytometry, or fluorescent reporters. Together, these techniques are beginning to elucidate cellular processes and pathways regulated by phosphorylation, in addition to global regulatory networks.     

Large‐scale phosphorylation mapping reveals the extent of tyrosine phosphorylation in Arabidopsis

Protein phosphorylation regulates a wide range of cellular processes. Here, we report the proteome‐wide mapping of in vivo phosphorylation sites in Arabidopsis by using complementary phosphopeptide enrichment techniques coupled with high‐accuracy mass spectrometry. Using unfractionated whole cell lysates of Arabidopsis, we identified 2597 phosphopeptides with 2172 high‐confidence, unique phosphorylation sites from 1346 proteins. The distribution of phosphoserine, phosphothreonine, and phosphotyrosine sites was 85.0, 10.7, and 4.3%. Although typical tyrosine‐specific protein kinases are absent in Arabidopsis, the proportion of phosphotyrosines among the phospho‐residues in Arabidopsis is similar to that in humans, where over 90 tyrosine‐specific protein kinases have been identified. In addition, the tyrosine phosphoproteome shows features distinct from those of the serine and threonine phosphoproteomes. Taken together, we highlight the extent and contribution of tyrosine phosphorylation in plants.     

Evolutionary Constraints of Phosphorylation in Eukaryotes,Prokaryotes, and Mitochondria

High accuracy mass spectrometry has proven to be a powerful technology for the large scale identification of serine/threonine/tyrosine phosphorylation in the living cell. However, despite many described phosphoproteomes, there has been no comparative study of the extent of phosphorylation and its evolutionary conservation in all domains of life. Here we analyze the results of phosphoproteomics studies performed with the same technology in a diverse set of organisms. For the most ancient organisms, the prokaryotes, only a few hundred proteins have been found to be phosphorylated. Applying the same technology to eukaryotic species resulted in the detection of thousands of phosphorylation events. Evolutionary analysis shows that prokaryotic phosphoproteins are preferentially conserved in all living organisms, whereas-site specific phosphorylation is not. Eukaryotic phosphosites are generally more conserved than their non-phosphorylated counterparts (with similar structural constraints) throughout the eukaryotic domain. Yeast and Caenorhabditis elegans are two exceptions, indicating that the majority of phosphorylation events evolved after the divergence of higher eukaryotes from yeast and reflecting the unusually large number of nematode-specific kinases. Mitochondria present an interesting intermediate link between the prokaryotic and eukaryotic domains. Applying the same technology to this organelle yielded 174 phosphorylation sites mapped to 74 proteins. Thus, the mitochondrial phosphoproteome is similarly sparse as the prokaryotic phosphoproteomes. As expected from the endosymbiotic theory, phosphorylated as well as non-phosphorylated mitochondrial proteins are significantly conserved in prokaryotes. However, mitochondrial phosphorylation sites are not conserved throughout prokaryotes, consistent with the notion that serine/threonine phosphorylation in prokaryotes occurred relatively recently in evolution. Thus, the phosphoproteome reflects major events in the evolution of life.Reversible protein phosphorylation on serines, threonines, and tyrosines plays a crucial role in regulating processes in all living organisms ranging from prokaryotes to eukaryotes (1). Traditionally, phosphorylation has been detected in single, purified proteins using in vitro assays. Recent advances in mass spectrometry (MS)-based proteomics now allow the identification of in vivo phosphorylation sites with high accuracy (2–7). On-line databases such as PhosphoSite (8), Phospho.ELM (9), and PHOSIDA1 (10) have collected and organized thousands of identified phosphosites. These databases as well as dedicated analysis environments such as NetworKIN (11, 12) offer and use contextual information including structural features, potential kinases, and conservation. They constitute resources that should allow the derivation of general patterns for phosphorylation events. Specifically, the recent availability of data for archaeal, prokaryotic, and diverse eukaryotic phosphoproteomes in these databases should enable investigation of the evolutionary history of this post-translational modification.Prokaryotes have two separate classes of phosphorylation events. Apart from the canonical histidine/aspartate phosphorylation, which has been studied for decades, serine/threonine/tyrosine phosphorylation is also present and has recently become amenable to analysis by MS (13). Bacterial phosphoproteins are involved in protein synthesis, carbohydrate metabolism, and the phosphoenolpyruvate-dependent phosphotransferase system. Recent phosphoproteomics studies of Bacillus subtilis, Escherichia coli, and Lactococcus lactis described around 100 phosphorylation sites on serine, threonine, and tyrosine in each of these species (13–15). Bacterial phosphorylation sites can change in response to environmental conditions (16).Interestingly, even archaea have serine/threonine and tyrosine phosphorylation. A recent study of Halobacterium salinarum described 75 serine/threonine/tyrosine phosphorylation sites on 62 proteins involved in a wide range of cellular processes including a variety of metabolic pathways (17).Although only a few hundred phosphorylation events have been found in prokaryotic species, similar experimental conditions and effort have yielded the detection of thousands of phosphorylation events in eukaryotes ranging from yeast to human (7, 18–21). Before the advent of large scale phosphoproteomics, serine/threonine/tyrosine phosphorylation has been estimated to affect one-third of all proteins (22). Recent large scale phosphoproteomics studies now suggest that more than half of all eukaryotic proteins are phosphorylated (23).A key event in evolution was the endosymbiosis of prokaryotes that enabled the development of a much more complex type of life, the eukaryotic cell. Analyses of mitochondrial genes suggest that the α-proteobacterium Rickettsia prowazekii is the endosymbiotic precursor leading to modern mitochondria (24). Almost all of the mitochondrial genes have migrated to the nuclear genome during subsequent evolution, and it is predicted that 10–15% of eukaryotic nuclear genes of organisms encode mitochondrial proteins (25).Thus, mitochondria with their unique evolutionary position between prokaryotes and eukaryotes form an interesting link for the evolutionary analysis of phosphorylation. Several studies investigated the mitochondrial phosphoproteome in different organisms using gel electrophoresis or specific enrichment methods coupled with mass spectrometry (26–28). Those studies established potential mitochondrial phosphoproteins. Three large scale studies based on affinity enrichment of phosphopeptides and mass spectrometry obtained direct experimental evidence of phosphorylation sites in mitochondria. Lee et al. (29) used a combination of different peptide enrichment strategies and found 80 phosphorylation sites of 48 different proteins from mouse liver. Very recently, a study by Deng et al. (30) characterized the murine cardiac mitochondrial mouse phosphoproteome, covering 236 phosphosites on 181 proteins. Investigating yeast, Reinders et al. (31) assigned 84 phosphorylation sites in 62 proteins.To enable comparative analysis of phosphoproteomes between all domains of life and mitochondria, here we experimentally determined a high accuracy mitochondrial mouse phosphoproteome based on technology conditions similar to those applied to the identification of prokaryotic and eukaryotic phosphoproteomes. We then performed a detailed evolutionary study of the conservation of the identified phosphoproteins and phosphorylation sites in prokaryotes and in eukaryotes. This allowed an initial comparison of the phosphoproteomes of prokaryotes, mitochondria, and eukaryotes.     

Integrative features of the yeast phosphoproteome and protein-protein interaction map

Following recent advances in high-throughput mass spectrometry (MS)-based proteomics, the numbers of identified phosphoproteins and their phosphosites have greatly increased in a wide variety of organisms. Although a critical role of phosphorylation is control of protein signaling, our understanding of the phosphoproteome remains limited. Here, we report unexpected, large-scale connections revealed between the phosphoproteome and protein interactome by integrative data-mining of yeast multi-omics data. First, new phosphoproteome data on yeast cells were obtained by MS-based proteomics and unified with publicly available yeast phosphoproteome data. This revealed that nearly 60% of ～6,000 yeast genes encode phosphoproteins. We mapped these unified phosphoproteome data on a yeast protein-protein interaction (PPI) network with other yeast multi-omics datasets containing information about proteome abundance, proteome disorders, literature-derived signaling reactomes, and in vitro substratomes of kinases. In the phospho-PPI, phosphoproteins had more interacting partners than nonphosphoproteins, implying that a large fraction of intracellular protein interaction patterns (including those of protein complex formation) is affected by reversible and alternative phosphorylation reactions. Although highly abundant or unstructured proteins have a high chance of both interacting with other proteins and being phosphorylated within cells, the difference between the number counts of interacting partners of phosphoproteins and nonphosphoproteins was significant independently of protein abundance and disorder level. Moreover, analysis of the phospho-PPI and yeast signaling reactome data suggested that co-phosphorylation of interacting proteins by single kinases is common within cells. These multi-omics analyses illuminate how wide-ranging intracellular phosphorylation events and the diversity of physical protein interactions are largely affected by each other.     

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.     

PACAP type I receptor transactivation is essential for IGF-1 receptor signalling and antiapoptotic activity in neurons

Insulin-like growth factor-1 (IGF-1) and pituitary adenylyl cyclase activating polypeptide (PACAP) are both potent neurotrophic and antiapoptotic factors, which exert their effects via phosphorylation cascades initiated by tyrosine kinase and G-protein-coupled receptors, respectively. Here, we have adapted a recently described phosphoproteomic approach to neuronal cultures to characterize the phosphoproteomes generated by these neurotrophic factors. Unexpectedly, IGF-1 and PACAP increased the phosphorylation state of a common set of proteins in neurons. Using PACAP type 1 receptor (PAC1R) null mice, we showed that IGF-1 transactivated PAC1Rs constitutively associated with IGF-1 receptors. This effect was mediated by Src family kinases, which induced PAC1R phosphorylation on tyrosine residues. PAC1R transactivation was responsible for a large fraction of the IGF-1-associated phosphoproteome and played a critical role in the antiapoptotic activity of IGF-1. Hence, in contrast to the general opinion that the trophic activity of IGF-1 is solely mediated by tyrosine kinase receptor-associated signalling, we show that it involves a more complex signalling network dependent on the PAC1 Gs-protein-coupled receptor in neurons.     

Ion mobility-resolved phosphoproteomics with dia-PASEF and short gradients

Mass spectrometry-based phosphoproteomics has identified >150,000 post-translational phosphorylation sites in the human proteome. To disentangle their functional relevance, complex experimental designs that require increased throughput are now coming into focus. Here, we apply dia-PASEF on a trapped ion mobility (TIMS) mass spectrometer to analyze the phosphoproteome of a human cancer cell line in short liquid chromatography gradients. At low sample amounts equivalent to ∼20 ug protein digest per analysis, we quantified over 13,000 phosphopeptides including ∼8700 class I phosphosites in 1 h without a spectral library. Decreasing the gradient time to 15 min yielded virtually identical coverage of the phosphoproteome, and with 7 min gradients we still quantified about 80% of the class I sites with a median coefficient of variation <10% in quadruplicates. We attribute this in part to the increased peak capacity, which effectively compensates for the higher peptide density per time unit in shorter gradients. Our data show a five-fold reduction in the number of co-isolated peptides with TIMS. In the most extreme case, these were positional isomers of nearby phosphosites that remained unresolved with fast liquid chromatography. In summary, our study demonstrates how key features of dia-PASEF translate to phosphoproteomics.     

Ser/Thr/Tyr phosphoproteome analysis of pathogenic and non‐pathogenic Pseudomonas species

Protein phosphorylation on serine, threonine, and tyrosine is well established as a crucial regulatory posttranslational modification in eukaryotes. With the recent whole‐genome sequencing projects reporting the presence of serine/threonine kinases and two‐component proteins both in prokaryotes and eukaryotes, the importance of protein phosphorylation in archaea and bacteria is gaining acceptance. While conventional biochemical methods failed to obtain a snapshot of the bacterial phosphoproteomes, advances in MS methods have paved the way for in‐depth mapping of phosphorylation sites. Here, we present phosphoproteomes of two ecologically diverse non‐enteric Gram‐negative bacteria captured by a nanoLC‐MS‐based approach combined with a novel phosphoenrichment method. While the phosphoproteome data from the two species are not very similar, the results reflect high similarity to the previously published dataset in terms of the pathways the phosphoproteins belong to. This study additionally provides evidence to prior observations that protein phosphorylation is common in bacteria. Notably, phosphoproteins identified in Pseudomonas aeruginosa belong to motility, transport, and pathogenicity pathways that are critical for survival and virulence. We report, for the first time, that motility regulator A, probably acting via the novel secondary messenger cyclic diguanylate monophosphate, significantly affects protein phosphorylation in Pseudomonas putida.     

Revealing the dynamics of the 20 S proteasome phosphoproteome: a combined CID and electron transfer dissociation approach

The 20 S proteasomes play a critical role in intracellular homeostasis and stress response. Their function is tuned by covalent modifications, such as phosphorylation. In this study, we performed a comprehensive characterization of the phosphoproteome for the 20 S proteasome complexes in both the murine heart and liver. A platform combining parallel approaches in differential sample fractionation (SDS-PAGE, IEF, and two-dimensional electrophoresis), enzymatic digestion (trypsin and chymotrypsin), phosphopeptide enrichment (TiO(2)), and peptide fragmentation (CID and electron transfer dissociation (ETD)) has proven to be essential for identifying low abundance phosphopeptides. As a result, a total of 52 phosphorylation identifications were made in mammalian tissues; 44 of them were novel. These identifications include single (serine, threonine, and tyrosine) and dual phosphorylation peptides. 34 phosphopeptides were identified by CID; 10 phosphopeptides, including a key modification on the catalytically essential beta5 subunit, were identified only by ETD; eight phosphopeptides were shared identifications by both CID and ETD. Besides the commonly shared phosphorylation sites, unique sites were detected in the murine heart and liver, documenting variances in phosphorylation between tissues within the proteasome populations. Furthermore the biological significance of these 20 S phosphoproteomes was evaluated. The role of cAMP-dependent protein kinase A (PKA) to modulate these phosphoproteomes was examined. Using a proteomics approach, many of the cardiac and hepatic 20 S subunits were found to be substrate targets of PKA. Incubation of the intact 20 S proteasome complexes with active PKA enhanced phosphorylation in both existing PKA phosphorylation sites as well as novel sites in these 20 S subunits. Furthermore treatment with active PKA significantly elevated all three peptidase activities (beta1 caspase-like, beta2 trypsin-like, and beta5 chymotrypsin-like), demonstrating a functional role of PKA in governing these 20 S phosphoproteomes.     

The Ser/Thr/Tyr phosphoproteome of Lactococcus lactis IL1403 reveals multiply phosphorylated proteins

Recent phosphoproteomics studies of several bacterial species have firmly established protein phosphorylation on Ser/Thr/Tyr residues as a PTM in bacteria. In particular, our recent reports on the Ser/Thr/Tyr phosphoproteomes of bacterial model organisms Bacillus subtilis and Escherichia coli detected over 100 phosphorylation events in each of the bacterial species. Here we extend our analyses to Lactococcus lactis, a lactic acid bacterium widely employed by the food industry, in which protein phosphorylation at Ser/Thr/Tyr residues was barely studied at all. Despite the lack of almost any prior evidence of Ser/Thr/Tyr protein phosphorylation in L. lactis, we identified a phosphoproteome of a size comparable to that of E. coli and B. subtilis, with 73 phosphorylation sites distributed over 63 different proteins. The presence of several multiply phosphorylated proteins, as well as over-representation of phosphothreonines seems to be the distinguishing features of the L. lactis phosphoproteome. Evolutionary comparison and the conservation of phosphorylation sites in different bacterial organisms indicate that a majority of the detected phosphorylation sites are species-specific, and therefore have probably co-evolved with the adaptation of the bacterial species to their present-day ecological niches.     

Evolution of protein phosphorylation for distinct functional modules in vertebrate genomes

Recent publications have revealed that the evolution of phosphosites is influenced by the local protein structures and whether the phosphosites have characterized functions or not. With knowledge of the wide functional range of phosphorylation, we attempted to clarify whether the evolutionary conservation of phosphosites is different among distinct functional modules. We grouped the phosphosites in the human genome into the modules according to the functional categories of KEGG (Kyoto Encyclopedia of Genes and Genomes) and investigated their evolutionary conservation in vertebrate genomes from mouse to zebrafish. We have found that the phosphosites in the vertebrate-specific functional modules (VFMs), such as cellular signaling processes and responses to stimuli, are evolutionarily more conserved than those in the basic functional modules (BFMs), such as metabolic and genetic processes. The phosphosites in the VFMs are also significantly more conserved than their flanking regions, whereas those in the BFMs are not. These results hold for both serine/threonine and tyrosine residues, although the fraction of phosphorylated tyrosine residues is increased in the VFMs. Moreover, the difference in the evolutionary conservation of the phosphosites between the VFMs and BFMs could not be explained by the difference in the local protein structures. There is also a higher fraction of phosphosites with known functions in the VFMs than BFMs. Based on these findings, we have concluded that protein phosphorylation may play more dominant roles for the VFMs than BFMs during the vertebrate evolution. As phosphorylation is a quite rapid biological reaction, the VFMs that quickly respond to outer stimuli and inner signals might heavily depend on this regulatory mechanism. Our results imply that phosphorylation may have an essential role in the evolution of vertebrates.