Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.13.3 (histidine kinase)
2,405 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

OmpR is a transcriptional activator for the ompF and ompC genes of Escherichia coli. Its phosphorylation is mediated by a transmembrane sensory-receptor protein, EnvZ, and is essential for transcriptional activation. In a previous study, when the aspartic acid residue at position 55, the putative phosphorylation site, was replaced with glutamine (D55Q), ompF and ompC expression were completely lost. In this study two pseudorevertants of the D55Q mutation were isolated and identified to be the replacement of threonine at position 83 with alanine (T83A) and glycine at position 94 with serine (G94S). The revertant OmpRs no longer responded to EnvZ function when ompF and ompC expression were examined. The purified D55Q-T83A OmpR was unable to be phosphorylated by EnvZ in vitro. The role of EnvZ as an osmosensor for the environmentally regulated expression of OmpF and OmpC has been indicated in previous studies. The isolation of seemingly EnvZ-independent OmpR revertants in this study, however, made it possible to examine the osmolarity-regulated expression of OmpF and OmpC in the absence of effects exerted by EnvZ. We found that the expression of OmpF and OmpC supported by these revertant OmpRs was clearly regulated in accordance with the change in osmolarity of the growth media. These results indicate that another EnvZ-independent mechanism(s) may also contribute to the regulated expression of the ompF and ompC genes.
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PMID:Intramolecular second-site revertants to the phosphorylation site mutation in OmpR, a kinase-dependent transcriptional activator in Escherichia coli. 164 88

The Tar-EnvZ hybrid molecule (Taz1) is an inner membrane transducer that activates OmpR, a transcriptional activator for porin gene expression (ompC), in response to an aspartic acid signal. Signal transduction by Taz1 most likely involves a phosphorylated Taz1 intermediate that donates its phosphate to OmpR. Phosphorylated OmpR has already been implicated in transcriptional activation of porin genes. Using a cell-free system containing Taz1-enriched membrane fractions, we have examined the phosphorylation properties of Taz1 and the stimulatory effects of divalent and monovalent ions. Highest activation of Taz1 phosphorylation was observed with CaCl2, and its stimulation could be observed with as low as 60 microM of CaCl2. Phosphorylated Taz1 could readily donate its phosphate group to OmpR in the presence of calcium. CaCl2 was also able to enhance phosphorylation of intact membrane-bound EnvZ and a cytoplasmic fragment of EnvZ lacking the receptor and transmembrane domains. These results indicate that the site for CaCl2 stimulation is within the cytoplasmic region of EnvZ and probably involves an enhanced rate of EnvZ phosphorylation.
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PMID:Ca2(+)-enhanced phosphorylation of a chimeric protein kinase involved with bacterial signal transduction. 185 Apr 14

Phosphorylation of OmpR, a transcription activator for ompF and ompC expression, is essential for its function and has been shown to be mediated in vitro by EnvZ, a transmembrane sensory receptor protein. On the basis of the three-dimensional structure of CheY which has an extensive sequence similarity with OmpR, three aspartic residues, D11, D12, and D55, of OmpR are considered to form a triacidic pocket serving as the phosphorylation center. When these aspartic acid residues were replaced with asparagine (D11N) or glutamine (D12Q and D55Q), ompF and ompC expression was almost completely blocked. Two pseudorevertants of the D11N mutation were isolated: one of them is a mutation in EnvZ (G240E), and the other is a mutation in OmpR (S48F). The envZ mutation (G240E) by itself was found to confer a phenotype very similar to that of the well known envZ11 mutation (T247R), suggesting that EnvZ (G240E) is an elevated kinase for OmpR. Consistent with this notion, EnvZ (T247R) was also able to suppress the D11N mutation in OmpR. An in vitro phosphorylation study showed that while the wild-type OmpR was phosphorylated by EnvZ, the D11N OmpR was not. These results suggest that the D11N mutation alters OmpR conformation in such a way that OmpR is very poorly phosphorylated by EnvZ. On the basis of the in vivo and in vitro analysis, the mechanisms by which the G240E mutation in EnvZ and the S48F mutation in OmpR suppress the D11N mutation in OmpR are discussed.
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PMID:Suppression of a mutation in OmpR at the putative phosphorylation center by a mutant EnvZ protein in Escherichia coli. 198 53

In Escherichia coli the OmpR and EnvZ proteins regulate the expression of the outer membrane porin proteins OmpC and OmpF. EnvZ and OmpR belong to a family of sensor/effector protein pairs that control adaptation to a variety of environmental conditions. EnvZ acts as the sensor protein that phosphorylates OmpR, which in turn regulates porin gene expression. The level of phosphorylated OmpR appears to be a determining factor for ompC and ompF regulation. Phosphorylation of OmpR is considered to occur at one or more aspartic acid residues (Asp-11, Asp-12 and/or Asp-55) that are highly conserved among the effector proteins. In this report we biochemically characterized the aspartic acid residue(s) in OmpR that were phosphorylated by EnvZ. Reduction of aspartyl phosphate residues in the amino-terminal domain of OmpR with [3H]-NaBH4 indicated that Asp-55 was a primary site of modification. We further studied the role of the highly conserved aspartate residues by creating OmpR mutants having aspartate to alanine substitutions at positions 11 (D11A), 12 (D12A) and 55 (D55A). Studies of ompF and ompC expression as well as in vivo and in vitro phosphorylation experiments also demonstrated that while Asp-55 is the primary phosphate acceptor site in OmpR, Asp-11 may also serve as a phosphorylation site, particularly in the absence of Asp-55.
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PMID:Identification of a phosphorylation site and functional analysis of conserved aspartic acid residues of OmpR, a transcriptional activator for ompF and ompC in Escherichia coli. 793 54

In prokaryotes, in the absence of protein serine/threonine/tyrosine kinases, protein histidine kinases play a major role in signal transduction involved in cellular adaptation to various environmental changes and stresses. Histidine kinases phosphorylate their cognate response regulators at a specific aspartic acid residue with ATP in response to particular environmental signals. In this His-Asp phosphorelay signal transduction system, it is still unknown how the histidine kinase exerts its enzymatic function. Here we demonstrate that the cytoplasmic kinase domain of EnvZ, a transmembrane osmosensor of Escherichia coli can be further divided into two distinct functional subdomains: subdomain A [EnvZ(C). (223-289); 67 residues] and subdomain B [EnvZ(C).(290-450); 161 residues]. Subdomain A, with a high helical content, contains the autophosphorylation site, H-243, and forms a stable dimer having the recognition site for OmpR, the cognate response regulator of EnvZ. Subdomain B, an alpha/beta-protein, exists as a monomer. When mixed, the two subdomains reconstitute the kinase function to phosphorylate subdomain A at His-243 in the presence of ATP. Subsequently, the phosphorylated subdomain A is able to transfer its phosphate group to OmpR. The two-domain structure of this histidine kinase provides an insight into the structural arrangement of the enzyme and its transphosphorylation mechanism.
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PMID:Two-domain reconstitution of a functional protein histidine kinase. 961 80

The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively. Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase. We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress. Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p. In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression. As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p. The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p- and Prr1p-dependent transcription. We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast.
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PMID:Peroxide sensors for the fission yeast stress-activated mitogen-activated protein kinase pathway. 1117 24

OmpR and EnvZ comprise a two-component system that regulates the porin genes ompF and ompC in response to changes in osmolarity. EnvZ is autophosphorylated by intracellular ATP on a histidine residue, and it transfers the phosphoryl group to an aspartic acid residue of OmpR. EnvZ can also dephosphorylate phospho-OmpR (OmpR-P) to control the cellular level of OmpR-P. At low osmolarity, OmpR-P levels are low because of either low EnvZ kinase or high EnvZ phosphatase activities. At high osmolarity, OmpR-P is elevated. It has been proposed that EnvZ phosphatase is the activity that is regulated by osmolarity. OmpR is a two-domain response regulator; phosphorylation of OmpR increases its affinity for DNA, and DNA binding stimulates phosphorylation. The step that is affected by DNA depends upon the phosphodonor employed. In the present work, we have used fluorescence anisotropy and phosphotransfer assays to examine OmpR interactions with EnvZ. Our results indicate that phosphorylation greatly reduces the affinity of OmpR for the kinase, whereas DNA does not affect their interaction. The results presented cast serious doubts on the role of the EnvZ phosphatase in response to signaling in vivo.
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PMID:Phosphorylation alters the interaction of the response regulator OmpR with its sensor kinase EnvZ. 1179 22

Anaplasma phagocytophilum, the etiologic agent of human granulocytic anaplasmosis (HGA), has genes predicted to encode three sensor kinases, one of which is annotated PleC, and three response regulators, one of which is PleD. Prior to this study, the roles of PleC and PleD in the obligatory intracellular parasitism of A. phagocytophilum and their biochemical activities were unknown. The present study illustrates the relevance of these factors by demonstrating that both pleC and pleD were expressed in an HGA patient. During A. phagocytophilum development in human promyelocytic HL-60 cells, PleC and PleD were synchronously upregulated at the exponential growth stage and downregulated prior to extracellular release. A recombinant PleC kinase domain (rPleCHKD) has histidine kinase activity; no activity was observed when the conserved site of phosphorylation was replaced with alanine. A recombinant PleD (rPleD) has autokinase activity using phosphorylated rPleCHKD as the phosphoryl donor but not with two other recombinant histidine kinases. rPleCHKD could not serve as the phosphoryl donor for a mutant rPleD (with a conserved aspartic acid, the site of phosphorylation, replaced by alanine) or two other A. phagocytophilum recombinant response regulators. rPleD had diguanylate cyclase activity to generate cyclic (c) di-GMP from GTP in vitro. UV cross-linking of A. phagocytophilum lysate with c-di-[(32)P]GMP detected an approximately 47-kDa endogenous protein, presumably c-di-GMP downstream receptor. A new hydrophobic c-di-GMP derivative, 2'-O-di(tert-butyldimethylsilyl)-c-di-GMP, inhibited A. phagocytophilum infection in HL-60 cells. Our results suggest that the two-component PleC-PleD system is a diguanylate cyclase and that a c-di-GMP-receptor complex regulates A. phagocytophilum intracellular infection.
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PMID:The Anaplasma phagocytophilum PleC histidine kinase and PleD diguanylate cyclase two-component system and role of cyclic Di-GMP in host cell infection. 1904 57

Uropathogenic Escherichia coli (UPEC) can grow in environments with significantly elevated osmolarities, such as murine and human urinary tracts. OmpR is the response regulator part of a two-component OmpR-EnvZ regulatory system that responds to osmotic stresses. To determine the role of OmpR in UPEC survival, a DeltaompR mutant was created in the UPEC clinical isolate NU149. The DeltaompR mutant had a growth defect compared with the wild-type strain under osmotic stress conditions; this defect was complemented by the full-length ompR gene on a plasmid, but not with a mutant OmpR with an alanine substitution for aspartic acid at the phosphorylation site at position 55. Furthermore, the DeltaompR mutant displayed up to 2-log reduction in bacterial cell numbers in murine bladders and kidneys compared with wild-type bacteria after 5 days of infection. The ability of the bacteria to survive was restored to wild-type levels when the DeltaompR mutant strain was complemented with wild-type ompR, but not when the alanine-substituted ompR gene was used. This study has fulfilled molecular Koch's postulates by showing the pivotal role OmpR plays in UPEC survival within the murine urinary tract.
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PMID:Survival of uropathogenic Escherichia coli in the murine urinary tract is dependent on OmpR. 1938

Francisella tularensis subsp. tularensis is the etiologic agent of tularemia and has been designated a category A biothreat agent by the CDC. Tularemia is characterized by replication and dissemination within host phagocytes. Intramacrophage growth is dependent upon the regulation of Francisella pathogenicity island (FPI) virulence genes, which is poorly understood. Two-component regulatory systems (TCS) are widely employed by Gram-negative bacteria to monitor and respond to environmental signals. Virulent strains of F. tularensis subsp. tularensis are devoid of classical, tandemly arranged TCS genes, but orphaned members, such as that encoding the response regulator PmrA, have been identified. In the F. novicida model system, previous work has shown that a pmrA mutant shows decreased expression of FPI genes, is deficient for intramacrophage growth, and is avirulent in the mouse model. Here, we determine that phosphorylation aids PmrA binding to regulated promoters pmrA and the FPI-encoded pdpD, and KdpD is the histidine kinase primarily responsible for phosphorylation of PmrA at the aspartic acid at position 51 (D51). A strain expressing PmrA D51A retains some DNA binding but exhibits reduced expression of the PmrA regulon, is deficient for intramacrophage replication, and is attenuated in the mouse model. With regard to virulence gene induction, PmrA coprecipitates with the FPI transcription factors MglA and SspA, which bind RNA polymerase. Together, these data suggest a model of Francisella gene regulation that includes a TCS consisting of KdpD and PmrA. Once phosphorylated, PmrA binds to regulated gene promoters recruiting free or RNA polymerase-bound MglA and SspA to initiate FPI gene transcription.
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PMID:Regulation of virulence gene transcripts by the Francisella novicida orphan response regulator PmrA: role of phosphorylation and evidence of MglA/SspA interaction. 2023 8


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