Key words: Map kinase, RAS, histidine, cancer, signal transduction, Nm23, histone, G protein, phosphorylation.

Abstract

Intensive investigation of protein tyrosine, serine and threonine phosphorylation has lead to advances in signal transduction research and cancer treatment. This feature summarizes research on mammalian proteins exhibiting histidine phosphorylation. Histidine kinases are well known in prokaryotic and lower eukaryotic systems where they form the “two-component” signal transduction system. The relative invisibility of histidine phosphorylation in mammalian cells may result from technical obstacles such as its acid lability, which precludes detection in electrophoretic systems, amino acid sequencing, etc. Emerging data have identified mammalian histidine kinases for the Kinase suppressor of ras (Ksr), a scaffold molecule for the Map kinase pathway, as well as histone H4, Aldolase C and the b-subunit of heterotrimeric G proteins. Additional mammalian proteins of interest to signal transduction and cancer research exhibit histidine phosphorylation, including P-selectin, Annexin I and the 20S proteasome. Other candidate histidine phosphorylated proteins are identified. These data suggest the existence of another series of phosphorylation patterns in signal transduction.

INTRODUCTION - Histidine Phosphorylation

The signal transduction and cancer fields have intensively investigated the hydroxylamino acid protein kinases, which mediate substrate tyrosine, and serine/threonine phosphorylation (rev. in (1, 2)). Kinase inhibitors are now in clinical trial, and some show synergy with traditional chemotherapeutic agents (3-5). This review summarizes mounting evidence that another class of kinases exist in mammalian cells and contributes to biology, signaling and cancer: histidine kinases.

The mammalian histidine kinase field has been pioneered by the Matthews lab (6, 7). Unlike tyrosine and serine/threonine phosphoamino acids, phosphohistidine is formed by a N-P phosphoramidate bond (Fig. 1). Phosphorylation of the 1 and 3 nitrogens has been detected, and a 1,3 diphosphate form is theoretically possible (8). Histidine phosphorylation differs from that of tyrosine or serine/threonine in its high free energy of hydrolysis, which enables the formation of acyl phosphates, phosphoesters and phosphoanhydrides (8). It may also cause distinct changes in protein conformation and binding patterns, and set up high energy signal relays which are thought to incorporate multiple signals rapidly. Histidine kinases often differ from the hydroxylamino acid kinases by the formation of a histidine phosphorylated kinase intermediate, which then transfers the phosphate to the substrate protein. Evidence for the phosphorylation of substrate proteins on histidine, aspartate and serine are presented herein. Of those, phospho-histidine and -aspartate are high energy bonds, and could continue to pass a phosphate in a relay fashion. Estimates of the prevalence of protein phosphohistidine in mammalian cells have not been reported. However, in lower eukaryotes, approximately 6% of protein phosphorylation was on histidine, which was comparable to levels of other phosphoamino acids (6, 7). Histidine kinases are well known in prokaryotes and lower eukaryotes where they form the “two-component” signal transduction system, the major signal response cascade to environmental signals. This review will briefly summarize two-component pathways. While some mammalian homologs of two-component proteins exist, other mammalian histidine kinases share no structural or sequence similarity and appear to have evolved independently (8).

Histidine phosphorylation has remained obscure for several reasons. Phosphohistidine is acid-labile and is therefore undetectable under many commonly used experimental conditions, including SDS-PAGE fixation, TCA precipitation, partial acid hydrolysis for sequencing, etc. Phosphohistidine is often suggested by base-stable, acid-labile phosphorylation. However, this does not exclude phosphoarginine and -lysine, and phosphoamino acid analysis is diagnostic. The high bond energy may dictate a rapid reaction, which can be missed under conventional enzymatic time courses and protein:protein binding assays. Use of thiophosphorylated nucelotides in histidine kinase reactions has slowed down events to detectable levels.

This review describes the known mammalian histidine kinases and histidine phosphorylated proteins. The impact of histidine phosphorylation is not well understood and theories based on site directed mutagenesis and other types of data are presented. This list is supplemented by a separate description of proteins thought to exhibit histidine phosphorylation, for which the diagnostic phosphoamino acid analysis in mammalian cells has not been reported. Histidine phosphatases have been described in several reviews (7-9), and further define the signaling pathways. Even though more seems to remain unknown than known, this review identifies a diversity of histidine phsophorylated proteins which appear to contribute to many cellular functions, and cancer.

Histidine kinases in prokaryotic and lower eukaryotic “two-component” systems.

Many excellent reviews of two-component signaling are available (for example,(10-18)). Two-component systems constitute the primary signal transduction pathway for prokaryotes to respond to environmental changes. In their simplest form a sensor histidine kinase detects an environmental signal (chemotaxis, osmolarity, sporulation signals, etc.) and autophosphorylates on a histidine residue. Signaling is thought to bring a constitutively active ATP binding catalytic domain into proximity with a dimerization domain containing the histidine phosphorylation site; the functional sensor kinase is a homodimer (15). The phosphate is transferred to the aspartate of a response regulator protein, which often functions as a phosphorylation-dependent DNA-binding transcription factor (Fig. 2, top panel). A common variant of this scheme includes multiple histidine-aspartate-histidine-aspartate relays, which may permit the incorporation of multiple signals or increase the specificity of the response.

Several eukaryotic two-component pathways have been identified (Fig. 2, lower panel). Histidine kinases and response regulators often occur as two motifs present on a single hybrid protein. Where relays occur, the phosphate can be transferred to a third protein module, a histidine phosphotransfer (HPt)domain, before encountering another set of histidine kinases/response regulators or hybrid proteins. HPt modules are hypothesized to permit the integration of multiple signal relays. The most important distinction, however, rests in the output of the two-component signaling. Often these pathways tie to the Map kinases, Protein kinase A or other well known signaling proteins. Furthermore, in many of the well characterized eukaryotic two-component systems, signaling favors the maintenance of the “normal”, quiescent or differentiated state.

Mammalian Histidine Kinases

Nm23 Metastasis Suppressor. The Nm23 gene family was discovered on the basis of its reduced expression in highly metastatic melanoma cell lines, as compared to related, poorly metastatic cell lines (19, 20). Nm23 mRNA and protein levels have since been examined in human tumor cohorts (rev. in (21)). While most studies of breast, ovarian, hepatocellular, cervical and gastric carcinomas reported a significant association of low Nm23 expression with an aspect of aggressive disease (i.e., patient survival, lymph node metastases), Nm23 expression does not represent an independent prognostic or predictive factor. The operative definition of a metastasis suppressor gene is its ability to diminish metastases in vivo, without an effect on primary tumor size, when transfected into a metastatically competent, low-expressing cell line (22). Ten transfection studies confirm this function for nm23 (23-31). In vitro studies show reduced motility, invasion and anchorage-independent colonization, and increased differentiation following Nm23 overexpression.

Despite intense effort, the biochemical mechanism whereby Nm23 suppresses tumor metastasis remains unknown. Multiple biochemical functions have been ascribed to Nm23 proteins, including (1) interactions with numerous proteins; (2) nucleoside diphosphate kinase (NDP kinase) activity, in which Nm23 removes the terminal phosphate from a NTP to autophosphorylate on its histidine 118, then subsequently transfers the phosphate to a NDP to recreate a NTP; (3) DNA transactivation and nuclease activities; (4) serine or histidine protein kinase activities (rev. in (32)). Support for the histidine protein kinase activity as a possible mechanism of metastasis suppression derived from site-directed mutagenesis studies. Wild type and site-directed mutant constructs containing human nm23-H1 cDNA tethered to a constitutive promoter were transfected into human MDA-MB-435 breast carcinoma cells. In vitro motility assays using either a defined chemoattractant or to fetal calf serum were performed to correlate one aspect of nm23 metastasis suppressive capacity and protein sequence. Overexpression of wild type or S44A mutated Nm23-H1 suppressed motility in vitro, but mutation of Nm23-H1 proline 96 (P96S) or serine 120 (S120G) resulted in high levels of motility (33). A histidine 118 mutant of Nm23-H1 could not be transfected to produce viable cells. We and others subsequently produced recombinant wild type and site-directed mutant Nm23-H1 proteins and assayed them for functional activities. None of the NDP kinase activity, DNA transactivational activity, DNA cleavage activity or inhibition of Tiam GEF activity correlated with motility suppression ((32-36). Assays of histidine protein kinase activity, however, exhibited a general correlation with motility suppressive capacity (37-39).

Paul Wager and colleagues first described the histidine protein kinase activity of Nm23 proteins using ATP citrate lyase as a substrate (40). Recombinant, purified Nm23 protein was autophosphorylated on histidine 118 by incubation with g32P-ATP, and the free ATP removed by column chromatography. When autophosphorylated Nm23 was incubated with a lysate of PC12 cells, phosphorylation of a 120 kD band was detected by electrophoresis and autoradiography. Partial purification of the phosphorylated protein showed it to be ATP citrate lyase, which is the primary source of cytosolic Acetyl CoA, and is involved in lipogenesis and cholesterologenesis. Both the acid/base stability data and phosphoaminoacid analysis indicated that ATP citrate lyase was phosphorylated on a histidine residue.

Additionally, Wagner et. al. identified a 43 kDa protein from bovine brain membranes which was aspartate phosphorylated by g32P-ATP autophosphorylated Nm23 in vitro (38). The substrate protein failed to autophosphorylate. Importantly, both the P96S and S120G site-directed mutant recombinant Nm23 proteins, which failed to inhibit motility, were deficient in phosphorylation of the 43 kDa protein. Thus, a two-component like kinase pathway was identified in mammalian cells, and its activity correlated with metastasis suppression. Wagner subsequently identified the 43 kDa protein as Aldolase C and aspartate 319 as a site of phosphorylation (39). Aldolase A contains a glutamic acid at the equivalent position, indicating specificity in this pathway. Aldolase C is an interesting enzyme. A role for Aldolase C in signal transduction is suggested by its binding of Phospholipase D2 (41) and inositol 1,4,5-triphosphate (42), and the effect of aspartate phosphorylation on these functions will be of interest. We were able to confirm the correlation of Nm23 histidine protein kinase activity and metastasis suppression using several “ice bucket” substrates (37).

Objections to the histidine kinase activity of Nm23 have concerned possible autophosphorylation due to its NDP kinase activity. As a NDP kinase, Nm23 could phosphorylate contaminating ADP to generate ATP, which could then phosphorylate the substrate. The inability of Aldolase C to autophosphorylate unequivocally demonstrated a kinase activity. Other objections have focused on the small size of the active site pocket of Nm23 as mapped by x-ray crystallography. Several factors may be germane to this situation. First, the structure of Nm23 in solution may be different from that of its crystal structure. Second, the Drosophila homolog of nm23, awd, is involved in development and differentiation of the presumptive adult tissues post-metamorphosis (43, 44). In experiments where the null awd germ line was transfected by various nm23 cDNAs, only a small percentage of total adult protein levels was needed for full restoration of normal development and differentiation (45). These data suggest that a minor population of Nm23 protein may exist, with an altered structure/function that is biologically potent. Examples reported to date include a molten globule structure for Nm23 and a hybrid protein with GAPDH (46, 47).

We recently characterized another substrate for Nm23-H1 protein, in a histidine-serine phosphate transfer (48). Using the eukaryotic two-component systems, we investigated the Arabidopsis ethylene differentiation pathway (Fig. 2, lower panel). Most proteins in this pathway were hybrids, containing both the histidine kinase and aspartate receiver domains. However, the ERS ethylene receptor exhibited histidine kinase activity, and no evidence was found for an aspartate containing receiver interacting protein. Rather, ERS interacted with CTR1 to diminish Map kinase activation by an unknown mechanism (49). Thus, a new kind of “two component” reaction fed into a signaling pathway widely relevant to mammalian cells. Mammalian homologs of CTR1 were reported to include Raf-1 (49), but we noted a similar degree of homology to the Kinase suppressor or Ras (Ksr) protein (48). Antibodies to Nm23 co-immunoprecipitated Ksr from both 293T cells and human MDA-MB-435 breast carcinoma cells. Autophosphorylated recombinant Nm23-H1, when incubated with Ksr immunoprecipitated from transiently transfected 293 cells, phosphorylated full lenght and N-terminal Ksr, but not C-terminal Ksr or Raf-1. Ksr phosphorylation by Nm23-H1 was identified as serine upon phosphoamino acid analysis, and HPLC analysis of tryptic peptides of Ksr showed two peaks of phosphorylation. Site-directed mutation of Ksr confirmed Nm23-H1 phosphorylation on Ksr serine 392, a 14-3-3 binding site, and on serine 434 in conjunction with the serine 392 mutation. This pattern of phosphorylation is novel. Ksr is thought to be a scaffold molecule for the Erk Map kinase signaling pathway (rev. in (50))(Fig. 3). Scaffolds are thought to assemble components of a signal transduction pathway to increase efficiency, specificity, and intracellular co-localization. The effect of Nm23 phosphorylation of serines 392/434 on Ksr’s protein binding pattern, intracellular localization, and Map kinase activation will be of interest. Importantly, we reported that the ratio of activated (phosphorylated) Erk1/2 was diminished in nm23-H1 transfectants of the human MDA-MB-435 breast carcinoma cell line, as compared to vector-transfected controls. A transfectant overexpressing the histidine kinase deficient nm23-H1 P96S mutant exhibited high levels of activated Erk1/2 indicating that the histidine kinase activity of Nm23-H1 is required for inhibition of the Map kinase pathway.

Histone H4 Kinase. The compaction and three-dimensional organization of DNA is mediated in part by the formation of core nucleosomes, consisting of a DNA fragment surrounded by an octamer of two copies each of histones H2A, H2B, H3 and H4. Histones are subject to post-translational modifications including acetylation, methylation, phosphorylation, ribosylation and ubiquitination, which may affect transcription, DNA recombination, nucleosomal structure and DNA repair. Galasinski et al. (51) have reported that the kinetics of histones 2A, 3 and 4 post-translational modifications in response to a signaling event are distinct, suggesting specificity in their regulation. Histone H4 is modified by acetylation on lysines 5, 8, 12 and 16, phosphorylation on serine 1, and histidine phosphorylation.

Histone H4 histidine kinases have been identified in rat tissues, Walker 256 choriocarcinoma cells and porcine thymus; all require Mg++ and phosphorylate both H4 histidines, H18 and H75 (8). In the thymus, an in-gel renaturation and kinase assay identified four bands of potential histidine kinase activity in the 34-41 kDa range (52). H4 kinase activity in the liver has been linked to regeneration or development (8). An acid-labile kinase activity was increased in liver regeneration from acute or chronic damage, but not sham-operated liver. Increased kinase activity was also demonstrated in fetal liver, but not in proliferating hepatocytes during postnatal development. Unlike two-component histidine kinases and other mammalian histidine kinases, there was no evidence of a histine autophosphorylated form. However, the authors suggest that it may have been undetected for technical reasons.

The functional significance of histone H4 histidine phosphorylation is unknown. One theory proposes that H4 histidine phosphorylation contributes to nucleosome octamer stability. The imidazole ring of H75 has been shown to hydrogen bind with E90 of Histone 2B within the intact nucleosome core, which is thought to stabilize the nucleosome octamer. H18 resides in the N-terminal region of H4, which interacts with the H2A-2B dimer of an adjacent nucleosome particle and may stabilize particle-particle interactions.

Histidine 18 of histone H4 has also been identified as a target of nickel carcinogenesis, and it can be hypothesized that the phosphorylation status of this residue may alter this pathway. Carcinogenic nickel compounds enter airway cells, and the sites of major damage are the heterochromatic regions of chromosomes. Nickel was reported to bind DNA poorly, but bound histone H4 at H18 (53). Nickel has also been reported to decrease histone H4 acetylation on lysine 12 in mammalian cells, which may represent a positional effect of binding at H18 (54). If histidine phosphorylation affects histone H4 acetylation, it would be expected to impact gene expression patterns. Copper was also reported to bind H18 of histone H4, suggesting that this carcinogenic pathway may be more general (53).

Heterotrimeric G protein b subunit kinase. Heterotrimeric G proteins are coupled to hormone and neurotransmitter receptors. G proteins are composed of three subunits, the a subunit that binds GDP and the effector, and the b and g subunits, which dimerize. While the b-g dimer is necessary for G protein activation, its role is unclear. Wieland et al. reported that the b subunit of the retinal G protein transducin could be thiophosphorylated by g 35S-ATP (55); similar results were reported for b-subunits in human HL-60 cells (56), and multiple other mammalian tissues (57). Multiple methods of characterization of the b-subunit phosphorylation yielded phosphohistidine (57, 58). In a well characterized system, addition of the formyl peptide fMet-Leu-Phe (FMLP) increased the HL-60 cell G protein b subunit thiophosphorylation, suggesting that this phosphorylation event is linked to signaling (59). In islet cells of the pancreas, disruption of the a-b-g trimer structure abolished b-subunit histidine phosphorylation (60).

Incubation of the b-subunit alone or in combination with exogenous purified a-subunit of transducin failed to result in the phosphorylation of the b-subunit, but addition of a membrane fraction supported this event, suggesting the presence of a membrane associated histidine kinase (60). Recently, this group has characterized a histidine kinase from pancreatic b-cells, with an estimated molecular weight of 60-70 kDa (61). In pancreatic b-cells, the G protein activator mastoparan, but not its inactive mastoparan-17 analog, stimulated both histidine kinase activity and G protein b-subunit phosphorylation, again linking b-subunit histidine phosphorylation and G protein signaling ((61).

The functional role of b-subunit histidine phosphorylation is debated. A controversial hypothesis suggests that the high energy phosphate on histidine could be transferred to GDP on the a subunit to create GTP and G protein activation. This “transphosphorylation” could augment the typical GDP-GTP exchange mechanism for G protein activation, potentially to augment or maintain the activated state. Evidence for this hypothesis includes the formation of thiophosphorylated GTP from GDP-bound transducin a subunits, when incubated with thiophosphorylated b-subunits (55). Evidence against this hypothesis was reported by Hohenegger et. al. (62). While this group confirms histidine phosphorylation of human platelet membrane G protein b-subunits, use of compounds to covalently attach nucleotides to subunits failed to identify transphosphorylation of a-subunit. The authors suggest that GDP dissociates from G protein, is phosphorylated by NDP kinases to GTP, and binds and activates G proteins through the classic exchange mechanism. Similar arguments were convincingly made concerning the transphosphorylation of a small G protein by Nm23 (63, 64). In support of this hypothesis, Nederkoorn et al. (65) analyzed the a-b-g structure based on crystallographic data and presented a model for such transphosphorylation. This model details a stepwise series of proton transfers from the interior of the G protein to histidine 183 of the b-subunit using a Tyr-Arg-Tyr-Arg-Tyr pentad proton shuttle. The arrival of a proton at H183 would release the histidine phosphate through a SN2 reaction, transferring to the neighboring Arg a201. Subsequently movement to the a-subunit GDP would then generate GTP. However, the group did not comment on how the effect of covalent modifications of this protein would affect the chain of phosphate transfer, which could potentially resolve this disagreement. In conclusion, histidine phosphorylation of G protein b subunits is widely reported and linked to G protein signaling, but elucidation of its function will require further investigation.

Histidine phosphorylated proteins for which a kinase has not been identified.

Given the difficulty of detecting histidine phosphorylation, it is not surprising that histidine phosphorylated proteins have been identified ahead of the initiating kinases. Several such proteins are listed below, together with speculation on the functional role of phosphohistidine.

P-selectin. P-selectin is a leukocyte cell adhesion molecule best known for mediating the interactions of platelets and endothelial cells with monocytes and neutrophils. P-Selectin has been reported to play a critical role in the arrest of lymphoma cells (66). It is synthesized as an integral membrane protein in a-granules and is then transported to the cell surface. Fusion of the granules with the plasma membrane opens a portion of P-selectin previously inside the vesicle to the extracellular environment (67). Serine/threonine/tyrosine phosphorylation of P-selectin is also reported, and is thought to mediate intracellular trafficking. Platelet activation with either collagen or thrombin generates P-selectin histidine phosphorylation (68). The receptors for collagen (integrin) and thrombin (G protein coupled receptor) are distinct, with distinct downstream signaling effects, suggesting the generality of P-selectin phosphorylation. The kinetics of histidine phosphorylation and dephosphorylation are extremely rapid. Histidine phosphorylation has been reported in H771 and H773 of the cytoplasmic tail of P-selectin, although the kinase and its functional consequences are unknown.

Annexin I. Annexin I is a calcium and phospholipid binding protein thought to promote vesicle and membrane aggregation, which influences endocytosis and exocytosis (69). Many other functions have been suggested for this protein, and different patterns of expression associated with progression to cancer (70, 71). Annexin I was identified as a 37 kDa protein phosphorylated by incubation of tracheal epithelia with either ATP or GTP (72). Annexin I is known to be phosphorylated on N-terminal serine residues by Protein kinase C and on tyrosine residues in response to EGF receptor signaling. A transient histidine phosphorylation was localized to the C-terminal core (72). The core region of Annexin I contains Ca++ binding and phospholipid interactive elements of the protein, whose functions are modulated by N-terminal sequences. A conserved histidine residue at position 103 is thought to be a histidine phosphorylation site; H103 is located on the face of the Annexin I core that interacts with phospholipids. The authors hypothesize that phosphorylation-induced charge alterations on histidine may affect its membrane organization and aggregation functions. The conserved H103 is adjacent to a cAMP binding site, and cyclin AMP analogs inhibited histidine phosphorylation of Annexin I. Thus, Annexin I histidine phosphorylation may be linked to cAMP signalling. Autophosphorylation of Annexin I was not observed indicating the participation of an uncharacterized histidine kinase. The authors note the conservation of core sequences among the various other Annexins, suggesting the possibility that histidine phosphorylation may occur more widely within this family of proteins (72).

20S Proteasome. The 20S proteasome is a central enzyme in protein degradation which contributes to metabolism, cell cycle, immunogenicity, stress responses and cancer. The eukaryotic 20S proteasome consists of 28 subunits arranged in four heptameric rings around a central catalytic cavity. The 20S proteasome can complex with other subunits to form the 26S proteasome and PA28 complexes. Purified 20S proteasome from human lymphoblastoma cells autophosphorylated after incubation with g32P-ATP, and the phosphorylated form was identified as phosphohistidine based on alkali stability, acid lability and phosphoamino acid analysis (73). Separation of the 20S proteasome subunits diminished the histidine phosphorylation. The two subunits exhibiting histidine autophosphorylation were C5 and C8 (73). The C5 and C8 subunits do not participate directly in protein hydrolysis, but are thought to contribute to the active structure of the proteasome. C8 subunits, but not C5 subunits, can form homo-heptameric ring structures (74, 75). The C8 subunit also participates in binding of proteins fated for degradation such as p21 WAF1/CIP1 (76). C8 subunits, but not the 20S proteasome, have also been reported to interact with the alphaB-crystallin chaperone protein, suggesting that proteasome-independent interactions occur (77). Any of these functions would be of interest in terms of the effect of histidine phosphorylation.

Metabolic enzymes. In its forward reaction Succinyl CoA synthetase generates the citric acid cycle enzyme Syccinyl CoA synthetase, and in the reverse reaction it generates succinyl CoA for ketone body metabolism and synthesis of porphyrin. In the porcine heart, Histidine 259 of the a subunit is phosphorylated, and is reported to modify the stoichiometry of the binding site for the a subunit (78, 79). Fructose 2,6 bisphosphatase is a bifunctional enzyme which catalyzes synthesis and degradation of fructose 2,6 biphosphate. Rat liver enzyme has been shown to proceed via a histidine 258 phosphorylated intermediate, and mutation of histidine to alanine resulted in loss of activity. Other histidine residues may be involved as well (80, 81).

Acid-labile, Base-stable phosphorylated proteins:
Several additional proteins exhibit a phosphorylation pattern consistent with either histidine, lysine or arginine. Both 14-3-3t and Hsp70 were initially characterized by their ATPase activity (73, 82). This activity was reminiscent of the NDP kinase in forward-backward reactions using NDPs and NTPs, suggesting that a phosphohistidine intermediate could be involved. A mechanism of action similar to that of heterotrimeric G proteins is proposed for Hsp70. Proteins bind to a ADP-Hsp70 complex, resulting in exchange of ADP for ATP. The binding of ATP causes a conformational change which releases the bound protein. ATP is then hydrolyzed to ADP, beginning the cycle anew. Both the ADP-ATP exchange and ATPase portions of the sequence are regulated by co-chaperones, which are thought to interact with the N-terminus of Hsp70 and influence the peptide binding and release by the C-terminus. The authors propose that Hsp70 ADP-ATP exchange activity may be influenced by phosphorylation status (82).

It is interesting to note that, through evolution, a histidine residue has been critical to the function of co-chaperones for the Hsp70 family. In E. coli the DnaJ and GrpE accessory proteins are essential to Hsp70 function. DnaJ activates ATP hydrolysis. A conserved J domain containing the tripeptide HPD is essential for DnaJ activity. Mutation of the histidine in HPD abolishes ATPase stimulatory activity but not peptide binding (83, 84). Several mammalian proteins have been reported to bind and stimulate the ATPase activity of Hsp70 family members, MTJ1, ERDJ4, and secretory vesicle cysteine string proteins (Csps) (85-87). All require the histidine of the J domain HPD motif, suggesting a conserved function. It will be interesting to determine whether these conserved histidines exhibit phosphorylation or phosphotransfer, and whether this would impact Hsp70 function.

6. On the horizon?

The phospholipase D (PLD) superfamily is conserved through evolution. One or two copies of a motif containing a conserved histidine, HXKX4D, are present in most family members (88, 89). The superfamily includes phospholipase D, which is a key element in signal transduction. Phospholipase D hydrolyzes phosphatidylcholine to produce choline and phosphatidic acid, which in turn can be hydrolyzed to lysophosphatidic acid by Phospholipase A2 or to diacylglycerol by phosphatidic acid phosphohydrolase. These products affect the function of heterotrimeric G proteins, small G proteins, Protein kinase C, etc. Other members of the superfamily include cardiolipin synthases, phosphatidylserine synthases, pox viral envelope proteins and bacterial nucleases. In bacterial systems, mutation of the conserved histidine results in loss of enzymatic activity (88, 89). For the Nuc prokaryotic superfamily protein the phosphohistidine was demonstrated to mediate catalysis (89). The authors suggested that, given the conservation of this motif, phospholipase D may function similarly in other species. Mutation of the conserved histidine in human Phospholipase D resulted in loss of catalytic activity (90), but a formal demonstration that this is a site of histidine phosphorylation is lacking to date. Phospholipase D expression and activity is widely studied in cancer (91-98) and is the subject of therapeutic approaches.

Less well characterized but of interest is Elongation factor-2 kinase, which phosphorylates and inactivates eukaryotic translational elongation factor-2 (99). This enzyme does not exhibit homology to the known serine, threonine or tyrosine kinase families, and is capable of autophosphorylation. The predicted structure of its catalytic domain is similar to that of bacterial histidine kinases.

Conclusions

The field of mammalian histidine kinases is emerging and likely to contribute to our understanding of signaling and cancer. Evolutionary homologs of lower eukaryotic signaling pathways as well as new histidine kinase structures and functions appear operative. This field would benefit tremendously from a few technical advances. The production of an antibody to phosphohistidine would enable widespread easy experimentation. Recent approaches using peptides as substrates for purification assays, and in gel enzymatic assays appear useful. Like tyrosine kinase signaling, it will be important for translational development to identify which pathways are “dominant” for cancer development and control. Pharmaceutical companies have already identified histidine kinase inhibitors as a next generation approach to antibiotic resistant bacterial infection, and these technologies and agents may be readily transferable to the cancer field.

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Figure 1. Structure of phosphohistidines, with ionization at pH 8 (7).

Figure 2.Schematic of “two-component” signal transduction systems in prokaryotes and eukaryotes. Histidine kinases are denoted by boxes labeled H, aspartate receiver proteins marked D, and histidine phosphotransfer proteins labeled Hpt. Adapted from (10, 11).

Figure 3.Nm23 phosphorylation of Ksr via a histidine kinase pathway (top panel)(48). Schematic of Ksr as a scaffold for Erk Map kinase signal transduction (50). The Nm23-H1 phosphorylation sites are noted by stars, and include a 14-3-3 binding site as well as serine 434. The latter serine is a site of in vivo phosphorylation, but binding partners have not yet been identified.

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