MINIREVIEW Phosphorylation and dephosphorylation of histidine residues in proteins Susanne Klumpp and Josef Krieglstein Abteilung Biochemie und Institut fu ¨ r Pharmakologie und Toxikologie, Fachbereich Pharmazie, Philipps-Universita ¨ t Marburg, Germany Protein phosphorylation is a key mechanism for intracellular signal transduction in both prokaryotic and eukaryotic cells. Vertebrate proteins are prevalently phosphorylated on side chains that contain a hydroxyl group, such as serine, thre- onine and tyrosine residues. In the past decade, however, an increasing number of examples of histidine phosphorylation has been described. Because acid treatment of phospho- proteins during purification and detection o f phosphoamino acid analysis is routine, O-phosphomonoesters have been studied more often, and the existence of acid-labile phos- phates h as been largely o verlooked. The latter class of N-phosphoamidates may well be more widespread than is generally believed, even though the O-phosphates r emain th e major class in terms of quantity and extent of distribution in proteins. Phosphohistidine currently is estimated to be 10- to 100-fold more a bundant than phosphotyrosine, but less abundant than phosphoserine [Matthews, H.R. (1995) Pharmac. Ther. 67, 323–350.]. This minireview briefly summarizes the extensive knowledge of the key mechanisms and functions of phosphohistidine in bacteria. It also des- cribes the still limited, yet increasing, data from homologs of the bacterial two-component system. Finally, novel mechanisms of phosphorylation a nd dephosphorylation of histidine residues not related to the two-component system are d escribed. Keywords: signal transduction; histidine; kinase; phos- phatase; t wo-component system. CHEMISTRY, STABILITY AND DETECTION OF PHOSPHOHISTIDINE Whereas phosphorylation of serine, threonine or tyrosine results in the formation of a phosphoester linkage, phos- phorylation of histidine residues o ccurs on nitro gen atoms, producing a phosphoramidate bond. Phosphohistidines have a large standard free energy of hydrolysis making them the most unstable of any known phosphoamino acid (reviewed in [1]). This explains why phosphohistidines are utilized as enzyme intermediates, for example, in the catalytic mechanism of succinyl-CoA-synthetase or glucose-6-phosphatase. On the other hand, in histone H4, 1-phosphohistidine at residue 75 is relatively stable, having a half-life of 12 days at room temperature and pH 7.6 [2]. Those examples clearly demonstrate that the stability of phosphoramidate bonds in proteins is influenced by neigh- boring amino-acid residues and thus strongly varies depending on the nature o f t he protein. Accordingly, histidine phosphatases may be needed or not. Phosphohistidine mostly goes undetected in conven- tional s tudies of protein phosphorylation because of instability o f t he phosphate-nitrogen bond in acid solutions as used for routine phosphoamino acid analysis. I n contrast, phosphoserine, phosphothreonine and phospho- tyrosine resist acid treatment (Table 1) [ 3]. The detection of O-phosphates in acid hydrolysates of proteins is possible because the hydrolysis of the phosphomonoester bonds is considerably slower than the hydrolysis of p eptide bonds. Accordingly, no significant loss of phosphoryl groups is observed after precipitation of O-phosphorylated proteins by trichloroacetic acid as commonly used f or phosphatase activity measurements. Phosphoserine and phosphothreo- nine are labile under alkaline conditions unlike phospho- tyrosine, which provides a means to differentiate between these molecules. Phosphoramidates are extremely acid labile but relatively base stable, except for phosphoargi- nine. In contrast to the O -phosphates, all N -phosphates are unstable to neutral hydroxylamine, and pyridine also catalyzes their hydrolysis [3]. One- and two-dimensional thin layer chromatography procedures for separation of phosphohistidine from phos- phoserine, phosphothreonine and phosphotyrosine have been established previously [1,3]. Free phosphohistidine is rapidly hydrolyzed, a nd is thus not commercially available. It can be synthesized easily using polyhistidine/phosp horyl- chloride or L -histidine/potassium phosphoramidate [4]. Ion-exchange resins and reversed-phase column chroma- tography are useful to separate phosphohistidine from all other phosphoamino acids and also suitable to resolve t he isomers of phosphohistidine [1,4]. The simplest approach to detect phosphohistidine in 32 P-labeled prote ins is to fractionate the proteins o n a poly- acrylamide gel and blot the separated proteins to a poly (vinylidene difluoride) membrane. This membrane is then placed directly in 1 M KOH and incubated at 5 5 °Cfor2h, dried and autoradiographed [3,5]. Bands remaining repre- sent phosphotyrosine a nd ph osp hohistidine/phospholysine. Correspondence to S. Klumpp, Abteilung Biochemie, Fachbereich Pharmazie, Marbacher Weg 6, D-35032 Marburg, Germany. Fax: + 49 6421 282 6645, Tel.: + 49 6421 282 6646, E-mail: klumpp@mailer.uni-marburg.de (Received 6 August 200 1, revised 2 1 September 2001, accepted 26 September 2001) Eur. J. Biochem. 269, 1067–1071 (2002) Ó FEBS 2002 Additional treatment of the membrane with 6 M HCl at 55 °C for 2 h leaves only phosphotyrosine. Diethyl pyro- carbonate is the most frequently used histidine modifying reagent p reventing s ubsequent phosphorylation of histidine residues. Screening for proteins containing N -linked phosphate with antibodies to phosphohistidine is not yet possible. Attempts t o prepare antibodies to derivatives o r conjugates of phosphohistidine have not been successful. However, many of the commercially available anti-phosphotyrosine Ig also recognize phosphohistidine. PHOSPHOHISTIDINE IN BACTERIA: THE TWO-COMPONENT SYSTEMS In prokaryo tic signaling, the p redominant phosphorylation scheme is referred to as Ôtwo-componentÕ system [6,7]. It frequently is involved in linking an extracellular stimulus such a s changing osmolarity, oxyg en, n itrogen or phospho- rus levels to gene regulating events, but also affects differentiation and other functions, such as chemotaxis. Two-component systems are abundant in most eubacte- ria, in which they typically constitute at least 1% of encoded proteins. The Escherichia c oli genome encodes 62 two- component proteins. Two-component systems are present in both Gram p ositive and Gram negative b acteria. In addition to housekeeping functions, they also control expression of toxins and o ther proteins i mportant for pathogenesis. The two-component signal transduction pathway consists of a sensor that is connected to a regulator through histidine phosphorylation and a subsequent phosphotransfer event to aspartate. The response t ime is q uite fast. The two- component system comprises several c haracteristic domains usually structured on two conserved proteins: a histidine kinase/sensor and a response regulator that are phosphor- ylated at histidine and aspartate residues, respectively (Fig. 1). Stimuli, detected by a sensor domain of the histidine kinase, regulate histid ine k inase a ctivities. The histidine kinase c atalyzes an ATP-dependent trans-auto- phosphorylation reaction i n which one subunit o f the dimer phosphorylates a specific histidine residue within the other Table 1. Chemical stability of phosphorylated amino acids. +, stable phosphoamino acid; –, labile phosphoamino acid. Stability in Nature of phosphoamino acid Acid Alkali O-Phosphates Phosphoserine + – Phosphothreonine + ± Phophotyrosine + + N-Phosphates Phosphoarginine – – Phosphohistidine – + Phospholysine – + Acyl-Phosphate Phosphoaspartate – – Fig. 1. The two-component phosphotransfer scheme. This typically consists of a dimeric transmembrane sensor histidine kinase (component I) and a cytoplasmic response regulator (component II). However, there are variations, e.g. the cytosolic histidine kinases C heA, and multicomponent phosphorelay systems, which consist of even three proteins: a hybrid histidine kinase with an additional resp onse regulator domain at the C-terminus; a separate histidine-containing phosphotransfer protein that serves as a histidine phosphate intermediate; and the response regulator. The two-component h istidine kinase do main is a m odule o f % 250 amino acids that has four c onserved bloc ks of amino-acid sequences l ocated within the ATP -binding domain (N, G 1 , F an d G2). Similarly, the response regulator domain can be identified from th e number and spacing of conserved aspartate, l ysine, and hydrophobic r esid ues in a module of %120 amino acids. A bbreviation: TM, t ransmembrane. 1068 S. Klumpp and J. Krieglstein (Eur. J. Biochem. 269) Ó FEBS 2002 subunit resulting in a phosphoimidazole. The response regulator then catalyzes transfer of the phosphoryl group from the phosphohistidine to one of its o wn aspartate residues. Phosphorylation of the conserved regulatory domain of the response regulator activates an effector domain that elicits the specific output response, for example, change in flagella motion or change in transcription. Structures of the bacterial histidine protein kinase cata- lytic domains are unlike those of any p reviously character- ized serine-, threonine- or tyrosine kinase [8,9]. However, the histidine kinase structures are related to ATPase domains of the t ype I I t opoisomerase gyrase B and the chaperone Hsp90. A highly conserved glutamic acid residue is predic- ted to be involved in the catalytic mechanism of the ATPase enzymes. This glutamic acid is not present in the histidine kinase active site, which might e xplain why members of this superfamily function as kinases and others as ATPases. TWO-COMPONENT SYSTEMS IN YEAST, AMOEBA, FUNGI AND PLANT Although two-component pathways are common in bacteria, evidence for their e xistence in eukaryotes is scarce. Starting no more than a decade ago, the field of reversible histidine phosphorylation i n e ukaryotes finally is beginning to emerge. By aligning members of the bacterial two- component histidine kinase f amily, oligonucleotide p rimers were designed to amplify t he kinase domain. This approach proved successful to clone homologs to the bacterial histidine kinases and response regulators in yeast, amoeba, fungi and plants. The best documented linkage between prokaryotic and eukaryotic two-component signal transduction mechanisms is for the osmoregulation system in yeast. The SLN1 and SSK1 proteins of Saccharomyces cerevisiae have sequence similarities to both the histidine kinase and the response regulator p roteins from bacteria, respectively [10,11]. Gen- etic analysis of site-directed mutants indicates that SLN1/ SSK1 act in the same manner as do its bacterial counter- parts, i.e. through f ormation of a phosphohistidyl enzyme followed by phosphotransfer to an aspartyl residue on its response regulator. Interestingly, this histidine protein kinase from yeast is part of a signaling cascade in which traditional eukaryotic mitogen-activated protein k inases participate as downstream elements (HOG1 MAPK cascade). The gene dokA from the slime mold Dictyostelium discoideum codes for a homolog of the bacterial hybrid histidine kinase family which is defined by the presence of conserved a mino-acid sequence motifs corresponding to an N-terminal receptor domain, a central histidine kinase and a C-terminal response regulator [12]. DokA mutants are deficient in the osmoregulatory pathway, resulting in premature cell death of this amoeba under high osmotic stress. The predicted protein sequence of the gene nik-1 from the fungus Neurospora crassa also shares homology with both the kinase and response regulator modules of two-com- ponent signaling proteins [13]. Deletion studies revealed that Nik-1 is involved in p roper h yphal development. In p lants, the simple gas ethylene (C 2 H 4 ) serves as a hormone with profound effects on g rowth and development. The C-terminal half of the ethylene response protein ETR1 in the p lant Arab idopsis thalia na is similar i n sequence to both components o f t he prokaryotic family of signal transducers known as the two-component system [14,15]. Similar to SLN1, ETR1 also i s involved in a MAPK cascade. One has to bear in mind, however, t hat the statement of dealing with analogs of the prokaryotic two-component systems so far is based on homology cloning resulting in amino-acid similarity and based on functional studies employing deletion/mutations. Histidine phosphorylation of the isolated or recombinant proteins, as extensively performed for the bacterial histidine kinase CheA, has not been studied. So far, only one protein histidine kinase has been purified from eukaryotes on the basis of activity measurements. The enzyme was isolated from S. cerevisiae [16]. HISTIDINE PHOSPHORYLATION AND DEPHOSPHORYLATION IN MAMMALIAN SYSTEMS Until now, two-component proteins have not been identi- fied in a nimals and a re not encoded by worm, fly or human genomes. It therefore has been suggested that the building blocks of the two-component systems in lower organisms may provide a target for the development of antibiotics directed against both fungal a nd bacterial pathogens i n vertebrates. Two eukaryotic genes have been discovered whose predicted products exhibit limited structural homology to the bacterial histidine protein kinases, however, functional homology c ould not be dem onstrated. The gene which encodes branched-chain a-ketoacid dehydrogenase kinase (BCKDH kinase) w as cloned in 1992 [17]. The sequence of this enzyme shows no resemblance to any e ukaryotic protein kinase. The closest homologs to BCKDH kinase are found among the histidine protein kinases of bacteria. Direct evidence for histidine phosphorylation of BCKDH kinase still is lacking. In contrast, BCKDH kinase auto- phosphorylates on serine and phosphorylates its physiolo- gical substrate, branched-chain a-ketoacid dehydrogenase, on a p air of serine residues a s well. Pyruvate dehydrogenase kinase from rats is an other e xample o f a protein with weak resemblance to histidine p rotein kinases from bacteria. As described for BCKDH kinase, pyruvate dehydrogenase kinase phosphorylates proteins on hydroxyl amino acids. Although there is no evidence for the existence of two- component systems in vertebrates yet, there are numerous reports on histidine phosphorylation i n v ertebrate p roteins. The description o f phosphohistidine in mammals started in the 1970s when Smith’s group [18] studied a rat nuclear protein kinase that phosphorylates histone H4 on histidine. In the 1990s, Motojima & Goto [19] and Hedge & Das [20] were studying the proteins p36 and p38, respectively. The proteins may be identical: both proteins are p hosphorylated in response to the presence of Ras protein and guanine nucleotides, and the phosphorylation occurs on a histidine residue. Noiman & Shaul [21] developed a protocol for rapid detection of histidine phosphoproteins in cellular crude extracts. Running the phosphorylation reactions in the presence of 5 m M EDTA (instead of Mg 2+ ) exclusively results in histidine phosphorylation. In 1995 , a nov el pathway for activation-dependent signal transduction in vertebrates was introduced [22]. A ligand- induced cascade g enerates phosphohistidine in platelets. Ó FEBS 2002 Histidine phosphorylation (Eur. J. Biochem. 269) 1069 In addition to well known phosphorylation on serine, threonine and tyrosine residues, thrombin and collagen lead to the phosphorylation of histidine in the cytoplasmic tail of P-selectin. The appearance and disappearance of phos- phohistidine on P-selectin are very rapid. The next finding of an intracellular signaling system involving histidine phosphorylation in mammals appeared in 2000 [23]. A 37-kDa protein from airway epithelia is phosphorylated on histidine and was identified as annexin I. It is a member of a family of Ca 2+ -dependent phospholipid- binding proteins whose phosphorylation is regulated by the chloride-ion concentration. The failure of annexin I to autophosphorylate i ndicates t hat i t is a substrate for a distinct yet undiscovered histidine kinase. Histidine i s an i mportant catalytic residue in many enzymes. In some cases, the histidine is covalently modified during the reaction. In a further subset, the modified form is phosphohistidine. Such phosphohistidine intermediates are not discussed in this article. DEPHOSPHORYLATION OF PHOSPHOHISTIDINE In prokaryotes Many bacterial two-component histidine k inases are bifunctional, having both kinase activity (acting on histi- dine) and phosphatase activity (acting on phosphoaspar- tate). Response regulators are the targets for the histidine kinase phosphatases. In the case of EnvZ, the osmosensor in E. coli and its cognate r esponse regulator OmpR [24], it has been suggested that OmpR-P is regulated by the OmpR-P phosphatase activity, whereas the OmpR kinase activity is maintained at a c onstant le vel. Similarly, in the CheA chemotaxis system, dephosphorylation of the phospho- CheY response regulator is modulated by the CheZ phosphatase. The SixA protein from E. coli is a prokaryotic phos- phohistidine phosphatase discovered recently [25]. SixA plays a role in d own-regulation of the Arc B-to-ArcA phosphorelay under certain anaerobic respiratory condi- tions. This histidine phosphatase activity is directed towards the phosphotransmitter domain of the bacterial hybrid histidine kinase. SixA consists of 161 amino acids and has an arginine-histidine-glycine signature at the N-terminus, which presumably functions as a nucleophilic phosp hoac- ceptor. In mammals Because a mammalian histidine kinase is not available yet, all published studies of protein histidine phosphatases in vertebrates so far have been carried out with histone H4 phosphorylated by the histidine kinase purified from yeast. Among the classical eukaryo tic protein phosphatases, protein tyrosine p hosphatases and the serine/threonine phosphatase type 2B (calcineurin) do not dephosphorylate H4 phosphorylated on histidine. The serine/threonine protein phosphatases type 1, t ype 2 A a nd type 2C, in contrast, a re highly active against phosphohistidine in histone H4 (reviewed in [1]). Intriguingly, none of the serine/threonine phosphatases acting on histidine phosphor- ylated histone, hydrolyze phosphohistidine using the bacterial histidine kinase CheA autophosphorylated on histidine 48 as a substrate [26]. In 2000, SixA was the first bacterial histidine phosphatase identified implicated in the histidine to aspartate phospho- relay (see above [25]). In the meantime, the first mammalian protein histidine phosphatase has also b een discovered [26]. Both enzymes are o f low apparent molecular m ass (17 and 14 k Da, respectively), hence similar in size to eukaryotic low molecular mass protein tyrosine phosphatases. But the proteins are c learly distinct. Vertebrate p rotein histidine phosphatase (PHP1) was isolated from rabbit liver. Its amino-acid sequence shows no resemblance to any phos- phatase described so far. Furthermore, inhibitors of phos- phatases acting on phosphoserine, phosphothreonine and phosphotyrosine residues h ad no effect. Protein histidine phosphatase is present in a variety of species from human to nematodes, but absent in bacteria. It is highly expressed throughout different tissues. ATP-citrate lyase known to undergo autophosphorylation on histidine [27] as well as external phosphorylation on histidine via nucleoside diphosphate kinase [28], is t he first vertebrate substrate identified for protein histidine phosphatase [26]. SUMMARY Knowledge of the phosphorylation and dephosphorylation of histidine residues in bacterial proteins is vast. In contrast, very little is known about the reversible phos- phorylation of histidine in vertebrates. We have recently learned that the dogma of histidine phosphorylation/ dephosphorylation in bacteria on the one hand vs. serine/ threonine and tyrosine phosphorylation/dephosphorylation in vertebrates on the other, is no longer realistic [29]. 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