Focus on phosphohistidine

Summary. Phosphohistidine has been identified as an enzymic intermediate in numerous biochemical reactions and plays a functional role in many regulatory pathways. Unlike the phosphoester bond of its cousins (phosphoserine, phosphothreonine and phosphotyrosine), the phosphoramidate (P–N) bond of phosphohistidine has a high ΔG° of hydrolysis and is unstable under acidic conditions. This acid-lability has meant that the study of protein histidine phosphorylation and the associated protein kinases has been slower to progress than other protein phosphorylation studies. Histidine phosphorylation is a crucial component of cell signalling in prokaryotes and lower eukaryotes. It is also now becoming widely reported in mammalian signalling pathways and implicated in certain human disease states. This review covers the chemistry of phosphohistidine in terms of its isomeric forms and chemical derivatives, how they can be synthesized, purified, identified and the relative stabilities of each of these forms. Furthermore, we highlight how this chemistry relates to the role of phosphohistidine in its various biological functions.     

Detection and analysis of protein histidine phosphorylation

Protein histidine phosphorylation is well established as an important part of signalling systems in bacteria, fungi and plants and there is growing evidence of its role in mammalian cell biology. Compared to phosphoserine, phosphothreonine and phosphotyrosine, phosphohistidine is relatively labile, especially under the acidic conditions that were developed to analyse protein phosphorylation. In recent years, there has been an increasing impetus to develop specific methods for the analysis of histidine phosphorylation and assay of histidine kinase activity. Most recently attention has focussed on the application of mass spectrometry to this end. This review provides an overview of methods available for the detection and analysis of phosphohistidine in phosphoproteins, with particular emphasis on the application of mass spectrometric techniques.     

Mutational study of human phosphohistidine phosphatase: effect on enzymatic activity

Although protein histidine phosphorylation is estimated to account for about 6% of total protein phosphorylation in eukaryotes, knowledge on histidine phosphorylation and dephosphorylation is still limited. Recently, a few reports have appeared on a mammalian 14-kDa phosphohistidine phosphatase, also named protein histidine phosphatase. Molecular cloning of the protein has opened possibilities for exploring its properties and physiological role. In the present work, we have searched for potential active site residues in the human phosphohistidine phosphatase by point mutations of conserved histidine and arginine residues to alanine. When assayed by the phosphohistidine-containing peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide, mutants H53A and H102A showed no detectable activity. Compared to the wild-type recombinant enzyme, the specific activity of mutant R45A was decreased by one order of magnitude, that of mutant R78A was decreased by about 30%, while that of mutant H81A was essentially unchanged. These results will facilitate future studies of the reaction mechanism, substrate binding, and molecular structure of the phosphohistidine phosphatase.     

Mammalian histidine kinases

Protein phosphorylation is one of the most ubiquitous and important types of post-translational modification for the regulation of cell function. The importance of two-component histidine kinases in bacteria, fungi and plants has long been recognised. In mammals, the regulatory roles of serine/threonine and tyrosine kinases have attracted most attention. However, the existence of histidine kinases in mammalian cells has been known for many years, although little is still understood about their biological roles by comparison with the hydroxyamino acid kinases. In addition, with the exception of NDP kinase, other mammalian histidine kinases remain to be identified and characterised. NDP kinase is a multifunctional enzyme that appears to act as a protein histidine kinase and as such, to regulate the activation of some G-proteins. Histone H4 histidine kinase activity has been shown to correlate with cellular proliferation and there is evidence that it is an oncodevelopmental marker in liver. This review mainly concentrates on describing recent research on these two types of histidine kinase. Developments in methods for the detection and assay of histidine kinases, including mass spectrometric methods for the detection of phosphohistidines in proteins and in-gel kinase assays for histone H4 histidine kinases, are described. Little is known about inhibitors of mammalian histidine kinases, although there is much interest in two-component histidine kinase inhibitors as potential antibiotics. The inhibition of a histone H4 histidine kinase by genistein is described and that of two-component histidine kinase inhibitors of structurally-related mammalian protein kinases. In addition, recent findings concerning mammalian protein histidine phosphatases are briefly described.     

Identification and characterization of a mammalian 14-kDa phosphohistidine phosphatase.

Protein histidine phosphorylation in eukaryotes has been sparsely studied compared to protein serine/threonine and tyrosine phosphorylation. In an attempt to rectify this by probing porcine liver cytosol with the phosphohistidine-containing peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide (phosphopeptide I), we observed a phosphatase activity that was insensitive towards okadaic acid and EDTA. This suggested the existence of a phosphohistidine phosphatase different from protein phosphatase 1, 2A and 2C. A 1000-fold purification to apparent homogeneity gave a 14-kDa phosphatase with a specific activity of 3 micro mol.min-1.mg-1 at pH 7.5 with 7 micro m phosphopeptide I as substrate. Partial amino-acid sequence determination of the purified porcine enzyme by MS revealed similarity with a human sequence representing a human chromosome 9 gene of hitherto unknown function. Molecular cloning from a human embryonic kidney cell cDNA-library followed by expression and purification, yielded a protein with a molecular mass of 13 700 Da, and an EDTA-insensitive phosphohistidine phosphatase activity of 9 micro mol.min-1.mg-1 towards phosphopeptide I. No detectable activity was obtained towards a set of phosphoserine-, phosphothreonine-, and phosphotyrosine peptides. Northern blot analysis indicated that the human phosphohistidine phosphatase mRNA was present preferentially in heart and skeletal muscle. These results provide a new tool for studying eukaryotic histidine phosphorylation/dephosphorylation.     

蛋白组氨酸磷酸酶研究进展

主要概括磷酸酶的种类,原核细胞磷酸组氨酸生物功能及调控,哺乳动物组氨酸残基磷酸化、去磷酸化,以及组氨酸磷酸酶及其底物的最新研究进展. 信号转导在生长发育及细胞功能中起极其重要的作用. 无论在原核还是真核细胞,蛋白质磷酸化是细胞内信号转导的关键机制. 研究最多的可逆的真核蛋白磷酸化,主要发生在含有羟基的丝氨酸、苏氨酸和酪氨酸残基上. 不同的激酶和磷酸酶受不同机制的调节,而调节过程中出现的差异是人类很多疾病的潜在基础. 与大量有关羟基磷酸化氨基酸的报道相比,有关氨基磷酸化氨基酸的报道甚少. 据估计,自然界中存在的磷酸组氨酸比磷酸酪氨酸多10 ~ 100倍,但不如磷酸丝氨酸丰富. 虽然对脊椎动物蛋白质中存在磷酸组氨酸的认识可以追溯到20世纪60年代初, 但由于研究手段的限制,至今对脊椎动物蛋白组氨酸激酶及组氨酸磷酸酶的结构及功能知之甚少. 但是,近几年的研究有突破性的发现,克隆和重组表达哺乳动物组氨酸磷酸酶为研究氨基磷酸化氨基酸的生物功能翻开新的一章.     

Crystal structures of YBHB and YBCL from Escherichia coli, two bacterial homologues to a Raf kinase inhibitor protein.

In rat and human cells, RKIP (previously known as PEBP) was characterized as an inhibitor of the MEK phosphorylation by Raf-1. In Escherichia coli, the genes ybhb and ybcl possibly encode two RKIP homologues while in the genomes of other bacteria and archaebacteria other homologous genes of RKIP have been found. The parallel between the cellular signaling mechanisms in eukaryotes and prokaryotes suggests that these bacterial proteins could be involved in the regulation of protein phosphorylation by kinases as well. We first showed that the proteins YBHB and YBCL were present in the cytoplasm and periplasm of E. coli, respectively, after which we determined their crystallographic structures. These structures verify that YBHB and YBCL belong to the same structural family as mammalian RKIP/PEBP proteins. The general fold and the anion binding site of these proteins are extremely well conserved between mammals and bacteria and suggest functional similarities. However, the bacterial proteins also exhibit some specific structural features, like a substrate binding pocket formed by the dimerization interface and the absence of cis peptide bonds. This structural variety should correspond to the recognition of multiple cellular partners.     

First structure of a eukaryotic phosphohistidine phosphatase

Phosphatases are a diverse group of enzymes that regulate numerous cellular processes. Much of what is known relates to the tyrosine, threonine, and serine phosphatases, whereas the histidine phosphatases have not been studied as much. The structure of phosphohistidine phosphatase (PHPT1), the first identified eukaryotic-protein histidine phosphatase, has been determined to a resolution of 1.9A using multiple-wavelength anomalous dispersion methods. This enzyme can dephosphorylate a variety of proteins (e.g. ATP-citrate lyase and the beta-subunit of G proteins). A putative active site has been identified by its electrostatic character, ion binding, and conserved protein residues. Histidine 53 is proposed to play a major role in histidine dephosphorylation based on these observations and previous mutational studies. Models of peptide binding are discussed to suggest possible mechanisms for substrate recognition.     

Bacterial tyrosine kinases: evolution, biological function and structural insights

Reversible protein phosphorylation is a major mechanism in the regulation of fundamental signalling events in all living organisms. Bacteria have been shown to possess a versatile repertoire of protein kinases, including histidine and aspartic acid kinases, serine/threonine kinases, and more recently tyrosine and arginine kinases. Tyrosine phosphorylation is today recognized as a key regulatory device of bacterial physiology, linked to exopolysaccharide production, virulence, stress response and DNA metabolism. However, bacteria have evolved tyrosine kinases that share no resemblance with their eukaryotic counterparts and are unique in exploiting the ATP/GTP-binding Walker motif to catalyse autophosphorylation and substrate phosphorylation on tyrosine. These enzymes, named BY-kinases (for Bacterial tYrosine kinases), have been identified in a majority of sequenced bacterial genomes, and to date no orthologues have been found in Eukarya. The aim of this review was to present the most recent knowledge about BY-kinases by focusing primarily on their evolutionary origin, structural and functional aspects, and emerging regulatory potential based on recent bacterial phosphoproteomic studies.     

Bacterial signalling involving eukaryotic-type protein kinases

Protein Ser, Thr and Tyr kinases play essential roles in signal transduction in organisms ranging from yeast to mammals, where they regulate a variety of cellular activities. During the last few years, a number of genes that encode eukaryotic-type protein kinases have also been identified in four different bacterial species, suggesting that such enzymes are also widespread in prokaryotes. Although many of them have yet to be fully characterized, several studies indicate that eukaryotic-type protein kinases play important roles in regulating cellular activities of these bacteria, such as cell differentiation, pathogenicity and secondary metabolism. A model based on the possible coupling between two-component systems and eukaryotic-type protein kinases is proposed to explain the function of eukaryotic-type protein kinases in bacterial signalling in the light of studies in bacteria, as well as in plants and yeast. These two groups of eukaryotes possess signal-transduction pathways involving both two-component systems and eukaryotic protein kinases.     

Histidine phosphorylation in biological systems

Histidine phosphorylation is important in prokaryotes and occurs to the extent of 6% of total phosphorylation in eukaryotes. Nevertheless phosphohistidine residues are not normally observed in proteins due to rapid hydrolysis of the phosphoryl group under acidic conditions. Many rapid processes employ phosphohistidines, including the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), the bacterial two-component systems and reactions catalyzed by enzymes such as nucleoside diphosphate kinase and succinyl-CoA synthetase. In the PTS, the NMR structure of the phosphohistidine moiety of the phosphohistidine-containing protein was determined but no X-ray structures of phosphohistidine forms of PTS proteins have been elucidated. There have been crystal structures of a few phosphohistidine-containing proteins determined: nucleoside diphosphate kinase, succinyl-CoA synthetase, a cofactor-dependent phosphoglycerate mutase and the protein PAE2307 from the hyperthermophilic archaeon Pyrobaculum aerophilum. A common theme for these stable phosphohistidines is the occurrence of ion-pair hydrogen bonds (salt bridges) involving the non-phosphorylated nitrogen atom of the histidine imidazole ring with an acidic amino acid side chain.     

Reversible phosphorylation of histidine residues in vertebrate proteins

Knowledge on kinases and phosphatases acting on serine, threonine and tyrosine residues of vertebrate proteins is huge. These enzymes are still under intensive investigation at present. This is in sharp contrast to what is known about kinases and phosphatases acting on histidine, arginine, lysine and aspartate residues in vertebrate proteins. It also is in contrast to extensive studies of histidine/aspartate phosphorylation in prokaryotes. This minireview briefly summarizes what we have learned about the reversible phosphorylation of histidine residues in mammals. It is described how the field developed during 40 years of science. The article especially highlights the discovery of the first protein histidine phosphatase from vertebrates. Having identified and characterized a protein histidine phosphatase provides at least one desperately required tool to handle and study phosphorylation and dephosphorylation of histidine residues in vertebrates in more detail. Recent evidence even suggests an involvement of histidine phosphorylation in signal transduction.     

Regulatory proteins with a sense of direction: cell cycle signalling network in Caulobacter

Localization of kinases and other signalling molecules at discrete cellular locations is often an essential component of signal transduction in eukaryotes. Caulobacter crescentus is a small, single-celled bacterium that presumably lacks intracellular organelles. Yet in Caulobacter, the subcellular distribution of several two-component signal transduction proteins involved in the control of polar morphogenesis and cell cycle progression changes from a fairly dispersed distribution to a tight accumulation at one or both poles in a spatial and temporal pattern that is reproduced during each cell cycle. This cell cycle-dependent choreography suggests that similarly to what happens in eukaryotes, protein localization provides a means of modulating signal transduction in bacteria. Recent studies have provided important insights into the biological role and the mechanisms for the differential localization of these bacterial signalling proteins during the Caulobacter cell cycle.     

A eukaryotic type serine/threonine kinase and phosphatase in Streptococcus agalactiae reversibly phosphorylate an inorganic pyrophosphatase and affect growth,cell segregation,and virulence

Protein phosphorylation is essential for the regulation of cell growth, division, and differentiation in both prokaryotes and eukaryotes. Signal transduction in prokaryotes was previously thought to occur primarily by histidine kinases, involved in two-component signaling pathways. Lately, bacterial homologues of eukaryotic-type serine/threonine kinases and phosphatases have been found to be necessary for cellular functions such as growth, differentiation, pathogenicity, and secondary metabolism. The Gram-positive bacteria Streptococcus agalactiae (group B streptococci, GBS) is an important human pathogen. We have identified and characterized a eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Biochemical assays revealed that Stk1 has kinase activity and localizes to the membrane and that Stp1 is a soluble protein with manganese-dependent phosphatase activity on Stk1. Mutations in these genes exhibited pleiotropic effects on growth, virulence, and cell segregation of GBS. Complementation of these mutations restored the wild type phenotype linking these genes to the regulation of various cellular processes in GBS. In vitro phosphorylation of cell extracts from wild type and mutant strains revealed that Stk1 is essential for phosphorylation of six GBS proteins. We have identified the predominant endogenous substrate of both Stk1 and Stp1 as a manganese-dependent inorganic pyrophosphatase (PpaC) by liquid chromatography/tandem mass spectrometry. These results suggest that these eukaryotic-type enzymes regulate pyrophosphatase activity and other cellular functions of S. agalactiae.     

The serine/threonine/tyrosine phosphoproteome of the model bacterium Bacillus subtilis

Protein phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) is well established as a key regulatory posttranslational modification in eukaryotes, but little is known about its extent and function in prokaryotes. Although protein kinases and phosphatases have been predicted and identified in a variety of bacterial species, classical biochemical approaches have so far revealed only a few substrate proteins and even fewer phosphorylation sites. Bacillus subtilis is a model Gram-positive bacterium in which two-dimensional electrophoresis-based studies suggest that the Ser/Thr/Tyr phosphorylation should be present on more than a hundred proteins. However, so far only 16 phosphorylation sites on eight of its proteins have been determined, mostly in in vitro studies. Here we performed a global, gel-free, and site-specific analysis of the B. subtilis phosphoproteome using high accuracy mass spectrometry in combination with biochemical enrichment of phosphopeptides from digested cell lysates. We identified 103 unique phosphopeptides from 78 B. subtilis proteins and determined 78 phosphorylation sites: 54 on serine, 16 on threonine, and eight on tyrosine. Detected phosphoproteins are involved in a wide variety of metabolic processes but are enriched in carbohydrate metabolism. We report phosphorylation sites on almost all glycolytic and tricarboxylic acid cycle enzymes, several kinases, and members of the phosphoenolpyruvate-dependent phosphotransferase system. This significantly enlarged number of bacterial proteins known to be phosphorylated on Ser/Thr/Tyr residues strongly supports 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 analysis in bacteria.     

Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> Expresses Two Mitogen-Activated Protein Kinase Genes That Represent Distinct Protozoan Subfamilies

All eukaryotes express mitogen-activated protein kinases (MAPKs) that govern diverse cellular processes including proliferation, differentiation, and survival. Even though these proteins are highly conserved throughout nature, MAPKs from closely related species often possess distinct signature sequences, making them well suited as drug discovery targets. Based on the central amino acid in the TXY dual phosphorylation loop, mammalian MAPKs are classified as extracellular signal-regulated kinases (ERKs), c-Jun amino-terminal kinases (JNKs), or p38 stress-response MAPKs. The presence of MAPKs in nonmetazoan eukaryotes suggests significant evolutionary conservation of these important signalling pathways. We recently cloned a novel stress-response MAPK gene (tgMAPK1) from Toxoplasma gondii, an obligate intracellular human parasite that can cause life-threatening infections in immunocompromised patients, and we now present data on a second T. gondii MAPK gene (tgMAPK2) that we cloned. We show that tgMAPK1 and tgMAPK2 are members of two distinct and previously unknown protozoan MAPK subfamilies that we have named pzMAPKl/pzMAPK3 and pzMAPK2. Our phylogenetic analysis of a collection of protozoan and metazoan MAPK genes in relation to ERK8-like genes demonstrates that an ERK8-like family, which includes the pzMAPK2 subfamily, is represented across a large variety of eukaryotic kingdoms and is evolutionarily very distant from other MAPK families.     

Tyrosine phosphorylation: an emerging regulatory device of bacterial physiology

Tyrosine phosphorylation is a key device in numerous cellular functions in eukaryotes, but in bacteria this protein modification was largely ignored until the mid-1990s. The first conclusive evidence of bacterial tyrosine phosphorylation came only a decade ago. Since then, several tyrosine kinases exhibiting unexpected features have been identified in a variety of bacteria. These enzymes use homologues of Walker motifs of nucleotide-binding proteins for their catalytic mechanism, thus defining an idiosyncratic type of bacterial tyrosine kinases. Recently, bacterial tyrosine kinases have been found to phosphorylate an increasing list of endogenous protein substrates. This discovery contributes to the emerging picture that bacterial tyrosine phosphorylation is an important regulatory arsenal of bacterial physiology in addition to the classical serine/threonine kinases, and the 'two-component' and phosphotransferase systems.     

Protein histidine phosphorylation: increased stability of thiophosphohistidine.

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine and tyrosine have been extensively studied, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins has been discovered in prokaryotic organisms as well as in eukaryotic cells. The ability to biochemically analyze phosphohistidine residues in proteins, however, is severely hampered by its extreme lability under acidic conditions. In our studies we have found that by replacing the phosphate linked to the histidine residue with a thiophosphate, a phosphohistidine derivative with increased stability is formed. This allows the analysis of phosphohistidine-containing proteins by established biochemical techniques and will greatly aid in the investigation of the role of this posttranslational modification in cellular processes.