Protein Histidine Phosphorylation Research Articles

A pan-specific antibody for direct detection of protein histidine phosphorylation

Despite its importance in central metabolism and bacterial cell signaling, protein histidine phosphorylation has remained elusive with respect to its extent and functional roles in biological systems due to the lack of adequate research tools. We report the development of the first pan-pHis antibody using a stable phosphohistidine (pHis) mimetic as the hapten. This antibody was successfully used in ELISA, Western blot, dot blot, immunoprecipitation, and in detection and identification of histidine-phosphorylated proteins from native cell lysates when coupled with mass spectrometric analysis. We also observed that protein pHis levels in E. coli lysates depend on carbon source and nitrogen availability in the growth media. In particular, we found that pHis levels on PpsA are sensitive to nitrogen availability in vivo and that α-ketoglutarate (α-KG) inhibits phosphotransfer from phosphorylated phosphoenolpyruvate synthase (PpsA) to pyruvate. We expect this antibody to open opportunities for investigating other pHis-proteins and their functions.

Progress in Bioanalysis of Protein Histidine Phosphorylation

Acid-labile protein phosphorylation on histidine residues was hardly investigated by mass spectrometry-based technologies in the past. Artificial phosphorylation using potassium phosphoramidate now allows the generation of reference compounds and enables the systematic investigation of model substances generating confidence in experimental procedures.

Chasing Phosphohistidine, an Elusive Sibling in the Phosphoamino Acid Family

This year (2012) marks the 50th anniversary of the discovery of protein histidine phosphorylation. Phosphorylation of histidine (pHis) is now widely recognized as being critical to signaling processes in prokaryotes and lower eukaryotes. However, the modification is also becoming more widely reported in mammalian cellular processes and implicated in certain human disease states such as cancer and inflammation. Nonetheless, much remains to be understood about the role and extent of the modification in mammalian cell biology. Studying the functional role of pHis in signaling, either in vitro or in vivo, has proven devilishly hard, largely due to the chemical instability of the modification. As a consequence, we are currently handicapped by a chronic lack of chemical and biochemical tools with which to study histidine phosphorylation. Here, we discuss the challenges associated with studying the chemical biology of pHis and review recent progress that offers some hope that long-awaited biochemical reagents for studying this elusive posttranslational modification (PTM) might soon be available.

Protein histidine [de]phosphorylation in insulin secretion: abnormalities in models of impaired insulin secretion

In the majority of cell types, including the islet β-cell, transduction of extracellular signals involves ligand binding to a receptor, often followed by the activation G proteins and their effector modules. The islet β-cell is unusual in that glucose lacks an extracellular receptor. Instead, events consequent to glucose metabolism promote insulin secretion via the generation of diffusible second messengers and mobilization of calcium. A selective increase in intracellular calcium has been shown to regulate the phosphorylation status key islet proteins thereby facilitating insulin secretion. In addition to classical protein kinases [e.g., protein kinases A and C], recent studies from our laboratory have focused on the expression and function of various forms of NDPK/nm23-like histidine kinases in clonal β-cells, normal rodent, and human islets. Further, we recently reported localization of a cytosolic protein histidine phosphatase [PHP] in INS 832/13 cells, normal rat islets, and human islets. siRNA-mediated knock down of nm23-H1 and PHP in insulin-secreting INS 832/13 cells significantly attenuated glucose-induced insulin secretion. We also observed significant alterations in the expression and function of nm23-H1/PHP in β-cells chronically exposed to elevated levels of glucose and saturated fatty acids, such as palmitate (i.e., glucolipotoxicity). Similar changes were also noted in islets from the Goto-Kakizaki and Zucker Diabetic Fatty rats, two known models for type 2 diabetes. It is concluded that protein histidine phosphorylation-dephosphorylation cycles play novel regulatory roles in G protein-mediated physiological insulin secretion and that abnormalities in this signaling axis lead to impaired insulin secretion in glucolipotoxicity and type 2 diabetes.

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.

Solution structure and catalytic mechanism of human protein histidine phosphatase 1

Protein histidine phosphorylation exists widely in vertebrates, and it plays important roles in signal transduction and other cellular functions. However, knowledge about eukaryotic PHPT (protein histidine phosphatase) is still very limited. To date, only one vertebrate PHPT has been discovered, and two crystal structures of hPHPT1 (human PHPT1) have been solved. However, these two structures gave different ligand-binding sites and co-ordination patterns. In the present paper, we have solved the solution structures of hPHPT1 in both P(i)-free and P(i)-bound states. Through comparison of the structures, along with a mutagenesis study, we have determined the active site of hPHPT1. In contrast with previous results, our results indicate that the active site is located between helix alpha1 and loop L5. His(53) was identified to be the catalytic residue, and the NH groups of residues His(53), Ala(54) and Ala(96) and the OH group of Ser(94) should act as anchors of P(i) or substrate by forming H-bonds with P(i). On the basis of our results, a catalytic mechanism is proposed for hPHPT1: the imidazole ring of His(53) serves as a general base to activate a water molecule, and the activated water would attack the substrate as a nucleophile in the catalysis; the positively charged side chain of Lys(21) can help stabilize the transition state. No similar catalytic mechanism can be found in the EzCatDB database.

Immunohistochemical localization of phosphohistidine phosphatase PHPT1 in mouse and human tissues

Protein histidine phosphorylation accounts for about 6% of the total protein phosphorylation in eukaryotic cells; still details concerning histidine phosphorylation and dephosphorylation are limited. A mammalian 14-kDa phosphohistidine phosphatase, also denominated PHPT1, was found 6 years ago that provided a new tool in the study of phosphohistidine phosphorylation. The localization of PHPT1 mRNA by Northern blot analysis revealed high expression in heart and skeletal muscle. The main object of the present study was to determine the PHPT1 expression on protein level in mouse tissues in order to get further information on the physiological role of the enzyme. Tissue samples from adult mice and 14.5-day-old mouse embryos were processed for immunostaining using a PHPT1-specific polyclonal antibody. The same antibody was also provided to the Swedish human protein atlas project (HPR) (http://www.proteinatlas.org/index.php). The results from both studies were essentially consistent with the previously reported expression of mRNA of a few human tissues. In addition, several other tissues, including testis, displayed a high protein expression. A salient result of the present investigation was the ubiquitous expression of the PHPT1 protein and its high expression in continuously dividing epithelial cells.

Emerging roles for protein histidine phosphorylation in cellular signal transduction: lessons from the islet β‐cell

Protein phosphorylation represents one of the key regulatory events in physiological insulin secretion from the islet β-cell. In this context, several classes of protein kinases (e.g. calcium-, cyclic nucleotide- and phospholipid-dependent protein kinases and tyrosine kinases) have been characterized in the β-cell. The majority of phosphorylated amino acids identified include phosphoserine, phosphothreonine and phosphotyrosine. Protein histidine phosphorylation has been implicated in the prokaryotic and eukaryotic cellular signal transduction. Most notably, phoshohistidine accounts for 6% of total protein phosphorylation in eukaryotes, which makes it nearly 100-fold more abundant than phosphotyrosine, but less abundant than phosphoserine and phosphothreonine. However, very little is known about the number of proteins with phosphohistidines, since they are highly labile and are rapidly lost during phosphoamino acid identification under standard experimental conditions. The overall objectives of this review are to: (i) summarize the existing evidence indicating the subcellular distribution and characterization of various histidine kinases in the islet β-cell, (ii) describe evidence for functional regulation of these kinases by agonists of insulin secretion, (iii) present a working model to implicate novel regulatory roles for histidine kinases in the receptor-independent activation, by glucose, of G-proteins endogenous to the β-cell, (iv) summarize evidence supporting the localization of protein histidine phosphatases in the islet β-cell and (v) highlight experimental evidence suggesting potential defects in the histidine kinase signalling cascade in islets derived from the Goto-Kakizaki (GK) rat, a model for type 2 diabetes. Potential avenues for future research to further decipher regulatory roles for protein histidine phosphorylation in physiological insulin secretion are also discussed.

Mass spectrometric analysis of protein histidine phosphorylation

Protein histidine phosphorylation is now recognized as an important form of post-translational modification. The acid-lability of phosphohistidine has meant that this phosphorylation has not been as well studied as serine/threonine or tyrosine phosphorylation. We show that phosphohistidine and phosphohistidine-containing phosphopeptides derived from proteolytic digestion of phosphohistone H4 are detectable by ESI-MS. We also demonstrate reverse-phase HPLC separation of these phosphopeptides and their detection by MALDI-TOF-MS.

Focus on phosphohistidine

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 DeltaG degrees 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.

CFTR, chloride concentration and cell volume: could mammalian protein histidine phosphorylation play a latent role?

A considerable body of evidence indicates that the intracellular chloride concentration ([Cl-]i) is an important regulatory signal in epithelial ion transport. [Cl-]i regulates the open channel probability of sodium and chloride channels, the rate of chloride channel recycling to the apical membrane, cell volume homeostasis, the activity of sodium-coupled chloride entry pathways and G-protein activity. Cell volume goes awry in epithelial cells bearing mutant forms of the cystic fibrosis (CF) transmembrane conductance regulator protein (CFTR); however, the pathways that mediate this [Cl-]i effect at the apical membrane of polarized epithelia are unknown. Recently, we proposed a mechanism for the transduction of in vitro chloride concentration into a phosphorylation signal to proteins within the apical membrane of respiratory epithelia. Our studies show that an apically enriched plasma membrane fraction from a variety of species, including sheep, human and mouse airway, contains at least two membrane-bound protein kinases which exhibit a number of novel properties. Firstly, the phosphate is located on histidine residues within different families of proteins; one kinase(s) utilizes GTP rather than ATP as a phosphate donor and each kinase has its own unique profile of membrane protein phosphorylation (which itself varies with anion species). Secondly, both kinases mediate Cl- -dependent phosphorylation of an apical membrane protein around the established physiological values for [Cl-]i in airway epithelial cells ( approximately 40 mM); associated phosphatases also alter the net phosphoprotein profile of the apical membrane. These findings are reviewed and their potential roles explored in relation to the pathogenesis of CF using the control of cell volume as a model for disrupted cellular function in CF-affected epithelia.

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.

Differential regulation by fatty acids of protein histidine phosphorylation in rat pancreatic islets

Long-chain fatty acids (e.g. arachidonic acid) have been implicated in physiological control of insulin secretion. We previously reported histidine phosphorylation of at least two islet proteins (e.g., NDP kinase and the beta subunit of trimeric G-proteins), and suggested that such a signalling step may have regulatory roles in beta cell signal transduction, specifically at the level of G-protein activation. Since our earlier findings also indicated potential regulation by long-chain fatty acids of islet G-proteins, we undertook the current study to verify putative regulation, by fatty acids, of protein histidine phosphorylation of NDP kinase and Gbeta subunit in normal rat islets. The phosphoenzyme formation of NDP kinase was stimulated by various fatty acids in the following rank order: linoleic acid > arachidonic acid > oleic acid > palmitic acid = stearic acid = control. Furthermore, the catalytic activity of NDP kinase was stimulated by these fatty acids in the rank order of: oleic acid > arachidonic acid > linoleic acid > palmitic acid = stearic acid = control. Arachidonic acid methyl ester, an inactive analog of arachidonic acid, did not significantly affect either the phosphoenzyme formation or the catalytic activity of NDP kinase. Interestingly, arachidonic acid exerted dual effects on the histidine phosphorylation of beta subunit; it significantly stimulated the phosphorylation at 33 microM beyond which it was inhibitory. Together, these findings identify additional loci (e.g., NDP kinase and Gbeta subunit) at which unsaturated, but not saturated, fatty acids could exert their intracellular effects leading to exocytotic secretion of insulin.

Histone H4 histidine kinase displays the expression pattern of a liver oncodevelopmental marker

Protein phosphorylation is a vital process in the regulation of mammalian cell division and the protein kinases that catalyze the phosphorylation of proteins on serine, threonine and tyrosine residues have been well characterized. In contrast, little is known about the kinases involved in protein histidine phosphorylation, which have been described in various mammalian cells that are highly proliferative. Histone H4 histidine kinase (HHK) activity is highly active in regenerating rat liver. Using a novel and specific assay, we demonstrate that it is active in human fetal liver, essentially absent in adult liver and highly expressed in liver tumours. 'Normal' liver surrounding the HCC contains low to undetectable levels of HHK. In a rodent model of chronic liver injury that leads to HCC, its activity is induced. Two lines of evidence suggest that liver progenitor (oval) cells, which populate the liver at early stages following induction of liver damage are responsible for the increased activity. Purified oval cells, as well as cell lines established from primary cultures of oval cells express high levels of HHK. We propose that the pattern of expression of histone H4 histidine kinase activity justifies its classification as an oncodevelopmental marker and suggest it may be useful as a diagnostic marker for hepatocellular carcinoma as well for identifying preneoplastic lesions.

Regulatory roles for small G proteins in the pancreatic beta-cell: lessons from models of impaired insulin secretion.

Emerging evidence suggests that GTP-binding proteins (G proteins) play important regulatory roles in physiological insulin secretion from the islet beta-cell. Such conclusions were drawn primarily from experimental data derived through the use of specific inhibitors of G protein function. Data from gene depletion experiments appear to further substantiate key roles for these signaling proteins in the islet metabolism. The first part of this review will focus on findings supporting the hypothesis that activation of specific G proteins is essential for insulin secretion, including regulation of their function by posttranslational modifications at their COOH-terminal cysteines (e.g., isoprenylation). The second part will overview novel, non-receptor-dependent mechanism(s) whereby glucose might activate specific G proteins via protein histidine phosphorylation. The third section will review findings that appear to link abnormalities in the expression and/or functional activation of these key signaling proteins to impaired insulin secretion. It is hoped that this review will establish a basis for future research in this area of islet signal transduction, which presents a significant potential, not only in identifying key signaling proteins that are involved in physiological insulin secretion, but also in examining potential abnormalities in this signaling cascade that lead to islet dysfunction and onset of diabetes.

Defective protein histidine phosphorylation in islets from the Goto-Kakizaki diabetic rat.

We recently described novel regulatory roles for protein histidine phosphorylation of key islet proteins (e.g., nucleoside diphosphate kinase and succinyl thiokinase) in insulin secretion from the islet beta-cell (Kowluru A. Diabetologia 44: 89-94, 2001; Kowluru A, Tannous M, and Chen HQ. Arch Biochem Biophys 398: 160-169, 2002). In this context, we also characterized a novel, ATP- and GTP-sensitive protein histidine kinase in isolated beta-cells that catalyzed the histidine phosphorylation of islet (endogenous) proteins as well as exogenously added histone 4, and we implicated this kinase in the activation of islet endogenous G proteins (Kowluru A. Biochem Pharmacol 63: 2091-2100, 2002). In the present study, we describe abnormalities in ATP- or GTP-mediated histidine phosphorylation of nucleoside diphosphate kinase in islets derived from the Goto-Kakizaki (GK) rat, a model for non-insulin-dependent diabetes. Furthermore, we provide evidence for a marked reduction in the activities of ATP- or GTP-sensitive histidine kinases in GK rat islets. On the basis of these observations, we propose that alterations in protein histidine phosphorylation could contribute toward insulin-secretory abnormalities demonstrable in the diabetic islet.

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.

Identification and characterization of a novel protein histidine kinase in the islet β cell: evidence for its regulation by mastoparan, an activator of G-proteins and insulin secretion

Using insulin-secreting cells, we previously demonstrated that specific proteins associated with the cytosolic, secretory granule, and mitochondrial fractions undergo a novel type of phosphorylation on their histidine residues. Subsequently, we identified these proteins as the nucleoside diphosphate kinase (NDPK) [Kowluru and Metz, Biochemistry 1994;33:12495–503], the β subunit of trimeric GTP-binding proteins [Kowluru et al., Biochem J 1996;313:97–107], and the α subunit of succinyl-CoA synthetase [Kowluru, Diabetologia 2001;44:89–94], respectively. Since several other enzymes of intermediary metabolism (e.g. ATP-citrate lyase and glucose-6-phosphatase) also undergo histidine phosphorylation, these initial findings may have a more generalized significance to β cells. Herein, we characterized a novel protein histidine kinase in pancreatic β cells, and determined it to be acid- and heat-labile as well as alkali-resistant in its phosphorylation of histone 4. Such an activity was detected in normal rat islets, human islets, and clonal β (HIT-T15 and INS-1) cells, and could utilize either ATP or GTP as a phosphoryl donor (with K m values in the range of 60–100 μM). On a size-exclusion column, its molecular mass was estimated to be in the range of 60–70 kDa. It was stimulated by divalent cations (Mg 2+>Mn 2+>control=Ca 2+=Zn 2+=Co 2+), but was resistant to polyamines. It was inactivated by known in vitro inhibitors of protein histidine phosphorylation (e.g. UDP or cromoglycate). Mastoparan, a global activator of G-proteins and insulin secretion from isolated β cells, but not mastoparan-17, its inactive analog, stimulated histidine kinase activity and histidine phosphorylation of G β subunit and insulin secretion from isolated rat islets. These studies identify, for the first time, a protein kinase activity in the pancreatic β cell that does not act on traditional -Ser, -Tyr, or -Thr residues. They also establish a possible link between histidine kinase activity and G β phosphorylation in isolated β cells.

Protein histidine kinase.

AbstractProtein phosphorylation on histidine occurs in a large number of processes in prokaryotes and in an unknown number of processes in eukaryotes. The functions of protein phosphohistidine in prokaryotes are much more clearly understood (1,2. The methods presented here have been developed for the study of protein histidine phosphorylation in eukaryotes where phosphohistidine is found as an intermediate in some enzyme reactions and may also be involved in signaling mechanisms (3). In prokaryotes, two types of protein phosphorylation on histidine have been characterized in detail. The first, historically, is the phosphoenolpyruvate-sugar phosphotransferase system (4,5). In this system, phosphohistidine acts as a phosphate donor and the initial phosphate donor is phosphoenolpyruvate, so this is not a protein kinase system. The second prokaryotic system is the twocomponent regulatory system (1,2). The core of this system comprises three protein domains, which may or may not be on different polypeptides. The first domain is the protein kinase domain, the second is the histidine substrate for this kinase and the third contains an aspartate residue that removes the phosphate from phosphohistidine. Genes homologous to those of the two-component system have been identified in lower eukaryotes (1,6,7).KeywordsScintillation CounterHistidine KinaseElution TimeProtein Kinase DomainPhosphoamino AcidThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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.