PubMed Health⌕ Search

Biomedical subjects

M R Atkinson

Publications and source records attributed to M R Atkinson.

At least 19 recordsLinked to original sources
PubMed2000

Functional dissection of the dimerization and enzymatic activities of Escherichia coli nitrogen regulator II and their regulation by the PII protein.

The dimeric two-component system transmitter protein NRII (NtrB) of Escherichia coli, product of glnL (ntrB), controls transcription of nitrogen-regulated genes by catalyzing the phosphorylation and dephosphorylation of the transcription factor NRI (NtrC). Previous studies showed that the PII signal transduction protein inhibits the kinase activity of NRII and activates its phosphatase activity. We observed that PII greatly stimulated the NRII phosphatase activity under conditions where the cleavage of ATP was prevented, indicating that the phosphatase activity did not result simply from prevention of the antagonistic NRII kinase activity by PII. Rather, PII was an activator of the phosphatase activity. To study this regulation, we examined the dimerization and enzymatic activities of NRII and various polypeptides derived from NRII, and their regulation by PII. Our results were consistent with the hypothesis that NRII consists of three domains: an N-terminal domain found only in NRII proteins and two domains formed by the conserved transmitter module of NRII, the phosphotransferase/phosphatase/dimerization (central) domain and the kinase domain. All three domains were involved in regulating the kinase and phosphatase activities of NRII. The N-terminal domain was involved in intramolecular signal transduction, and controlled access to the NRII active site for the isolated dimeric central domain added in trans. The central domain was responsible for dimerization and the phosphotransferase and phosphatase activities of NRII, but the latter activity was weak in the isolated domain and was not regulated by PII. The C-terminal kinase domain was responsible for the kinase activity. The PII protein appeared to interact with the isolated transmitter module of NRII, and not with the N-terminal domain as previously thought, since PII dramatically increased the stoichiometry of autophosphorylation of the isolated transmitter module. However, the phosphatase activity of the transmitter module of NRII was low even in the presence of PII, suggesting that the N-terminal domain was necessary for the central domain to assume the conformation necessary for potent phosphatase activity. Also, PII significantly reduced the rate of transphosphorylation of the isolated central domain by the isolated kinase domain, suggesting that PII interacts directly with the kinase domain. We hypothesize that the binding of PII to the kinase domain of NRII results in an altered conformation that is transmitted to the central and N-terminal domains; this causes the central domain to assume the conformation with potent phosphatase activity.

ATP-Binding Cassette Transporters↗
PubMed2000

PII signal transduction proteins.

PII proteins, found in Bacteria, Archaea and plants, help coordinate carbon and nitrogen assimilation by regulating the activity of signal transduction enzymes in response to diverse signals. Recent studies of bacterial PII proteins have revealed a solution to the signal transduction problem of how to coordinate multiple receptors in response to diverse stimuli yet permit selective control of these receptors under various conditions and allow adaptation of the system as a whole to long-term stimulation.

Amino Acid Sequence↗
PubMed1999

Characterization of the GlnK protein of Escherichia coli.

The GlnK and PII signal transduction proteins are paralogues that play distinct roles in nitrogen regulation. Although cells lacking GlnK appear to have normal nitrogen regulation, in the absence of PII, the GlnK protein controls nitrogen assimilation by regulating the activities of the PII receptors glutamine synthetase adenylyltransferase (ATase) and the kinase/phosphatase nitrogen regulator II (NRII or NtrB), which controls transcription from nitrogen-regulated promoters. Here, the wild-type GlnK protein and two mutant forms of GlnK were purified, and their activities were compared with those of PII using purified components. GlnK and PII were observed to have unique properties. Both PII and GlnK were potent activators of the phosphatase activity of NRII, although PII was slightly more active. In contrast, PII was approximately 40-fold more potent than GlnK in the activation of the adenylylation of glutamine synthetase by ATase. While both GlnK and PII were readily uridylylated by the uridylyltransferase activity of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR), only PII approximately UMP was effectively deuridylylated by the UR activity of the UTase/UR. Finally, there were subtle differences in the regulation of GlnK activity by the small molecule effector 2-ketoglutarate compared with the regulation of PII activity by this effector. Altogether, these results suggest that GlnK is unlikely to play a significant role in the regulation of ATase in wild-type cells, and that the main role of GlnK may be to contribute to the regulation of NRII and perhaps additional, unknown receptors in nitrogen-starved cells. Also, the slow deuridylylation of GlnK approximately UMP by the UTase/UR suggests that rapid interconversion of GlnK between uridylylated and unmodified forms is not necessary for GlnK function. One mutant form of GlnK, containing the alteration R47W, was observed to lack specifically the ability to activate the NRII phosphatase in vitro; it was able to be uridylylated by the UTase/UR and to activate the adenylylation activity of ATase. Another mutant form of GlnK, containing the Y51N alteration at the site of uridylylation, was not uridylylated by the UTase/UR and was defective in the activation of both the NRII phosphatase activity and the ATase adenylylation activity.

Bacterial Proteins↗
PubMed1998

Role of the GlnK signal transduction protein in the regulation of nitrogen assimilation in Escherichia coli.

Two structurally similar but functionally distinct PII-like proteins, PII and GlnK, regulate nitrogen assimilation in Escherichia coli. Studies with cells indicated that both PII (the glnB product) and GlnK (the glnK product) acted through the kinase/phosphatase NRII [NtrB, the glnL (ntrB) product] to reduce transcription initiation from Ntr promoters, apparently by regulating the phosphorylation state of the transcriptional activator NRI-P (NtrC-P, the phosphorylated form of the glnG (ntrC) product). Both GlnK and PII also acted through adenylyltransferase (ATase, the glnE product) to regulate the adenylylation state of glutamine synthetase (GS). The activity of both GlnK and PII was regulated by the signal-transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR, glnD product). Our experiments indicate that either PII or GlnK could effectively regulate ATase, but that PII was required for the efficient regulation of NRII required to prevent expression of glnA, which encodes GS. Yet, GlnK also participated in regulation of NRII. Although cells that lack either PII or GlnK grew well, cells lacking both of these proteins were defective for growth on nitrogen-rich minimal media. This defect was alleviated by the loss of NRII, and was apparently due to unregulated expression of the Ntr regulon. Also, mutations in glnK, designated glnK*, were obtained as suppressors of the Ntr- phenotype of a double mutant lacking PII and the UTase/UR. These suppressors appeared to reduce, but not eliminate, the ability of GlnK to prevent Ntr gene expression by acting through NRII. We hypothesize that one role of GlnK is to regulate the expression of the level of NRI-P during conditions of severe nitrogen starvation, and by so doing to contribute to the regulation of certain Ntr genes.

ATP-Binding Cassette Transporters↗
PubMed1997

Expression of intercellular adhesion molecule-1 on macrophages in vitro as a marker of activation.

Macrophage activation is a major component of wound healing. It also determines the extent of inflammatory reactions and the response of the body to implanted materials. We have previously shown, using an in vitro model, that the extent of spreading of macrophages on different materials is a marker of activation, and that a soluble inducer has a dose-response effect on the secretion of cytokines in the culture medium. This work investigates the expression of three different cell surface markers [macrophages MAC-1, MAC-3 and intercellular adhesion molecule-1 (ICAM-1)] on macrophages in vitro using confocal microscopy and shows that ICAM-1 is also a marker of macrophage activation in this model. We observed increased amounts of ICAM-1 on activated macrophages compared to unactivated macrophages, whereas MAC-1 and MAC-3 were either expressed constitutively or demonstrated no quantitative change in expression after activation under the same experimental conditions. We also tested the expression of ICAM-1 with various concentrations of soluble inducers (lipopolysaccharide, 0.001, 0.01, 0.1, 1 and 10 micrograms ml-1. S-27609, 0.1, 0.25, 0.5, 1, 2 and 3 micrograms ml-1 and on a sheet of polylactic acid alone or in combination with soluble inducers. All doses of soluble inducers induced the expression of ICAM-1 on cells grown in glass chamber slides. The induction was not dose related but seemed to work rather in an on-off manner. There was no effect of material on ICAM-1 expression on the cell surface when no soluble inducer was added. This was similar to cytokine secretion, which was not induced by our material alone. When either lipopolysaccharide or S-27609 was used in combination with the material, there was an increase in the average measured intensity of ICAM-1. In this in vitro model, ICAM-1 staining as measured by confocal microscopy is a marker for macrophage activation. Our results suggest that the extent of macrophage activation as measured by ICAM-1 and by cytokine secretion is more sensitive to soluble inducers than to the action of the flat sheet of polylactic acid.

Aminoquinolines↗
PubMed1997

Cloning, characterisation and chromosomal assignment of the human adenosine A3 receptor (ADORA3) gene.

The gene for the inhibitory G-protein coupled human A3 adenosine receptor (ADORA3) was isolated and sequence analysis shows that the coding region is interrupted by a single intron of size 2.4 kb. The location of this intron in the second intracellular loop is conserved with respect to the A1, A2a and A2b adenosine receptor subtype genes. The ADORA3 gene was mapped to 1p13.3 by fluorescence in situ hybridisation. Northern blot studies show that the gene is widely expressed and is most abundant in brain and some endocrine tissues. We have mapped multiple transcription start sites in two cell lines and lung tissue by primer extension and 5' RACE (rapid amplification of cDNA ends). The ADORA3 gene promoter lacks CAAT and TATA boxes but has putative binding sites for multiple transcription factors. In contrast to the A1 adenosine receptor gene we find no evidence of alternate splicing in the 5' untranslated region of the ADORA3 gene.

Base Sequence↗
PubMed1997

Structure/function analysis of the PII signal transduction protein of Escherichia coli: genetic separation of interactions with protein receptors.

The PII protein, encoded by glnB, is known to interact with three bifunctional signal transducing enzymes (uridylyltransferase/uridylyl-removing enzyme, adenylyltransferase, and the kinase/phosphatase nitrogen regulator II [NRII or NtrB]) and three small-molecule effectors, glutamate, 2-ketoglutarate, and ATP. We constructed 15 conservative alterations of PII by site-specific mutagenesis of glnB and also isolated three random glnB mutants affecting nitrogen regulation. The abilities of the 18 altered PII proteins to interact with the PII receptors and the small-molecule effectors 2-ketoglutarate and ATP were examined by using purified components. Results with certain mutants suggested that the specificity for the various protein receptors was altered; other mutations affected the interaction with all three receptors and the small-molecule effectors to various extents. The apex of the large solvent-exposed T loop of the PII protein (P. D. Carr, E. Cheah, P. M. Suffolk, S. G. Vasudevan, N. E. Dixon, and D. L. Ollis, Acta Crytallogr. Sect. D 52:93-104, 1996), which includes the site of PII modification, was not required for the binding of small-molecule effectors but was necessary for the interaction with all three receptors. Mutations altering residues of this loop or affecting the nearby B loop of PII, which line a cleft between monomers in the trimeric PII, affected the interactions with protein receptors and the binding of small-molecule ligands. Thus, our results support the predictions made from structural studies that the exposed loops of PII and cleft formed at their interface are the sites of regulatory interactions.

Adenosine Triphosphate↗
PubMed1995

The Escherichia coli PII signal transduction protein is activated upon binding 2-ketoglutarate and ATP.

Nitrogen regulation of transcription in Escherichia coli requires sensation of the intracellular nitrogen status and control of the dephosphorylation of the transcriptional activator NRI-P. This dephosphorylation is catalyzed by the bifunctional kinase/phosphatase NRII in the presence of the dissociable PII protein. The ability of PII to stimulate the phosphatase activity of NRII is regulated by a signal transducing uridylyltransferase/uridylyl-removing enzyme (UTase/UR), which converts PII to PII-UMP under conditions of nitrogen starvation; this modification prevents PII from stimulating the dephosphorylation of NRI approximately P. We used purified components to examine the binding of small molecules to PII, the effect of small molecules on the stimulation of the NRII phosphatase activity by PII, the retention of PII on immobilized NRII, and the regulation of the uridylylation of PII by the UTase/UR enzyme. Our results indicate that PII is activated upon binding ATP and either 2-ketoglutarate or glutamate, and that the liganded form of PII binds much better to immobilized NRII. We also demonstrate that the concentration of glutamine required to inhibit the uridylyltransferase activity is independent of the concentration of 2-ketoglutarate present. We hypothesize that nitrogen sensation in E. coli involves the separate measurement of glutamine by the UTase/UR protein and 2-ketoglutarate by the PII protein.

Adenosine Triphosphate↗
PubMed1994

Reversible uridylylation of the Escherichia coli PII signal transduction protein regulates its ability to stimulate the dephosphorylation of the transcription factor nitrogen regulator I (NRI or NtrC).

We have reconstituted the signal transduction system responsible for the negative regulation of the transcription of the Escherichia coli glnA gene, encoding glutamine synthetase, by glutamine. This signal transduction system consists of four proteins: the transcription factor NRI (NtrC), which activates glnA transcription when it is phosphorylated, the kinase/phosphatase protein NRII (NtrB) that directly controls the extent of NRI phosphorylation, the PII signal transduction protein that controls the phosphatase activity of NRII, and the uridylyltransferase/uridylyl-removing (UTase/UR) enzyme that is regulated by glutamine and controls the activity of PII. In the reconstituted system, the removal of uridylyl groups from the PII protein, catalyzed by the UTase/UR protein in the presence of glutamine, resulted in the stimulation of NRI approximately P dephosphorylation. In contrast, the uridylylated form of the PII protein had no discernible effect on NRI phosphorylation. The uridylylation of the trimeric PII protein by the monomeric UTase/UR protein is a non-cooperative reaction in which the partially modified species accumulated and were readily observed. Partially modified PII trimers were partially active in stimulating the dephosphorylation of NRI approximately P. Thus, both the PII-UTase/UR and PII-NRII interactions display the continuous variability characteristic of rheostats as opposed to the binary variability characteristic of toggle switches.

Bacterial Proteins↗
PubMed1994

Effect of mutations in Escherichia coli glnL (ntrB), encoding nitrogen regulator II (NRII or NtrB), on the phosphatase activity involved in bacterial nitrogen regulation.

We examined the effects of mutations in glnL, encoding the signal-transducing kinase/phosphatase nitrogen regulator II (NRII), on the regulated phosphatase activity involved in nitrogen regulation. With wild-type NRII, this phosphatase activity was only observed in the presence of the signal transduction protein II (PII). Three different glnL mutations result in altered NRII proteins that had phosphatase activity in the absence of PII. The most active of these contained an alteration of the site of NRII autophosphorylation, histidine 139, to asparagine (H139N). The phosphatase activity of the NRII-H139N protein was further stimulated by the PII protein and by ATP. This suggests that the PII protein is not directly involved in a catalytic step of the regulated phosphatase activity but rather plays a regulatory role. We also measured the effect on the regulated phosphatase activity of alterations at conserved residues in the kinase/phosphatase domain of NRII and the effect of deleting the non-conserved N-terminal domain of NRII. For this we used fusion proteins containing the Escherichia coli maltose-binding protein (MBP) linked to the protein of interest. A protein consisting of MBP linked to wild-type NRII was a less active kinase than was wild-type NRII but in the presence of PII had wild-type phosphatase activity. A protein consisting of MBP linked to just the C-terminal domain of wild-type NRII had kinase activity but lacked phosphatase activity. Alterations at the highly conserved residues Asp-287, Gly-289, and Gly-291 in NRII affected both activities. A fusion of MBP to the NRII-H139N protein lacked kinase activity but had phosphatase activity in the absence of PII. Thus, while the kinase and phosphatase activities of NRII could be genetically separated, some of the highly conserved residues in the C-terminal domain of NRII (Asp-287, Gly-289, Gly-291) are apparently important for both activities.

Amino Acid Sequence↗
PubMed1994

The nitrogen-regulated Bacillus subtilis nrgAB operon encodes a membrane protein and a protein highly similar to the Escherichia coli glnB-encoded PII protein.

Expression of beta-galactosidase encoded by the nrg-29::Tn917-lacZ insertion increases 4,000-fold during nitrogen-limited growth (M.R. Atkinson and S. H. Fisher, J. Bacteriol. 173:23-27, 1991). The chromosomal DNA adjacent to the nrg-29::Tn917-lacZ insertion was cloned and sequenced. Analysis of the resulting nucleotide sequence revealed that the Tn917-lacZ transposon was inserted into the first gene of a dicistronic operon, nrgAB. The nrgA gene encodes a 43-kDa hydrophobic protein that is likely to be an integral membrane protein. The nrgB gene encodes a 13-kDa protein that has significant sequence similarity with the Escherichia coli glnB-encoded PII protein. Primer extension analysis revealed that the nrgAB operon is transcribed from a single promoter. The nucleotide sequence of this promoter has significant similarity with the -10 region, but not the -35 region, of the consensus sequence for Bacillus subtilis sigma A-dependent promoters.

Amino Acid Sequence↗
PubMed1994

Modulation of Bacillus subtilis catabolite repression by transition state regulatory protein AbrB.

The first enzyme of the Bacillus subtilis histidine-degradative (hut) pathway, histidase, was expressed at higher levels during the onset of the stationary growth phase in nutrient sporulation medium in early-blocked sporulation mutants (spo0A) than in wild-type strains. Histidase expression was also elevated in spo0A mutant cultures compared with wild-type cultures during the logarithmic growth phase in minimal medium containing slowly metabolized carbon sources. Histidase expression was not derepressed in spo0A abrB mutant cultures under these growth conditions, suggesting that the AbrB protein is responsible for the derepression of histidase synthesis seen in spo0A mutant cultures. spo0A mutants contain higher levels of the AbrB protein than do wild-type strains because the Spo0A protein represses AbrB expression. A direct correlation between the levels of abrB transcription and histidase expression was found in spo0A mutant cultures. The hutOCR2 operator, which is required for wild-type regulation of hut expression by catabolite repression, was also required for AbrB-dependent derepression of hut expression in spo0A mutants. Purified AbrB protein bound to the hutOCR2 operator in vitro, suggesting that AbrB protein alters hut expression by competing with the hut catabolite repressor protein for binding to the hutOCR2 site. During the logarithmic growth phase in media containing slowly metabolized carbon sources, the expression of several other enzymes subject to catabolite repression was elevated in spo0A mutants but not in spo0A abrB mutants. This suggests that the AbrB protein acts as a global modulator of catabolite repression during carbon-limited growth.

Arabinose↗
PubMed1994

Sensory components controlling bacterial nitrogen assimilation.

In enteric bacteria, the transcription of the Ntr regulon is regulated by a signal transduction system that measures and transmits information on the nitrogen status of the cell. Four of the components of this signal transduction apparatus have been previously identified, and the roles of these are known, to a first approximation, from studies with purified components. The sensor is a uridylyltransferase/uridylyl-removing enzyme (UTase/UR) that controls the uridylylation state of the PII protein. PII indirectly regulates the transcription of the Ntr regulon by acting through the kinase/phosphatase protein NRII. In the absence of unmodified PII, NRII autophosphorylates on a histidine residue, and these phosphoryl groups are transferred to the transcription factor NRI, resulting in the conversion of NRI to the form able to activate transcription. In the presence of PII and NRII, NRI approximately P is rapidly dephosphorylated, preventing the activation of Ntr transcription. This PII-dependent dephosphorylation of NRI approximately P is referred to as the regulated phosphatase activity. In this report, we describe improved methods for the purification of the UTase/UR and PII, and the crystallization of PII. We also present improved methods for the assay of the activities of the UTase/UR protein and PII. The results of our assays indicate that purified PII is effective in eliciting the regulated phosphatase activity, but does not affect the autophosphorylation of NRII or affect the transfer of phosphoryl groups from NRII approximately P to NRI. In addition, we demonstrate that the elicitation of the regulated phosphatase activity by PII is strongly dependent on the ratio of NRI approximately P to NRI, and that the isolated N-terminal domain of NRI, once phosphorylated, is dephosphorylated by the regulated phosphatase activity.

Amino Acid Sequence↗
PubMed1993

Activation of the Bacillus subtilis hut operon at the onset of stationary growth phase in nutrient sporulation medium results primarily from the relief of amino acid repression of histidine transport.

During growth of Bacillus subtilis in nutrient sporulation medium containing histidine (DSM-His medium), the expression of histidase, the first enzyme in the histidine-degradative pathway (hut), is derepressed 40- to 200-fold at the onset of stationary phase. To identify the gene products responsible for this regulation, histidase expression was examined in various hut regulatory mutants as well as in mutants defective in stationary-phase gene regulation. Histidase expression during growth in DSM-His medium was significantly altered only in a strain containing the hutC1 mutation. The hutC1 mutation allows the hut operon to be expressed in the absence of its inducer, histidine. During logarithmic growth in DSM-His medium, histidase levels were 25-fold higher in the HutC mutant than in wild-type cells. Moreover, histidase expression in the HutC mutant increased only four- to eightfold after the end of exponential growth in DSM-His medium. This suggests that histidine transport is reduced in wild-type cells during exponential growth in DSM-His medium and that this reduction is largely responsible for the repression of hut expression in cells growing logarithmically in this medium. Indeed, the rate of histidine uptake in DSM-His medium was fourfold lower in exponentially growing cells than in stationary-phase cells. The observation that the degradation of histidine is inhibited when B. subtilis is growing rapidly in medium containing a mixture of amino acids suggests that a hierarchy of amino acid utilization may be present in this bacterium.

Amino Acids↗
PubMed1993

Mutational analysis of the bacterial signal-transducing protein kinase/phosphatase nitrogen regulator II (NRII or NtrB).

The signal-transducing kinase/phosphatase nitrogen regulator II (NRII or NtrB) is required for the efficient positive and negative regulation of glnA, encoding glutamine synthetase, and the Ntr regulon in response to the availability of ammonia. Alteration of highly conserved residues within the kinase/phosphatase domain of NRII revealed that the positive and negative regulatory functions of NRII could be genetically separated and that negative regulation by NRII did not require the highly conserved His-139, Glu-140, Asn-248, Asp-287, Gly-289, Gly-291, Gly-313, or Gly-315 residue. These mutations affected the positive regulatory function of NRII to various extents. Certain substitutions at codons 139 and 140 resulted in mutant NRII proteins that were transdominant negative regulators of glnA and the Ntr regulon even in the absence of nitrogen limitation. In addition, we examined three small deletions near the 3' end of the gene encoding NRII; these resulted in altered proteins that retained the negative regulatory function but were defective to various extents in the positive regulatory function. A truncated NRII protein missing the C-terminal 59 codons because of a nonsense mutation at codon 291 lacked entirely the positive regulatory function but was a negative regulator of glnA even in the absence of nitrogen limitation. Thus, we have identified both point and deletion mutations that convert NRII into a negative regulator of glnA and the Ntr regulon irrespective of the nitrogen status of the cell.

Amino Acid Sequence↗
PubMed1993

Mechanism of autophosphorylation of Escherichia coli nitrogen regulator II (NRII or NtrB): trans-phosphorylation between subunits.

Nitrogen regulator II (NRII or NtrB) is a homodimeric signal-transducing protein kinase/phosphatase responsible for the transcriptional regulation of the Ntr regulon in Escherichia coli. NRII is a member of a large family of proteins that are part of the related two-component signal transduction systems. We studied the mechanism of NRII autophosphorylation by using purified components. Alteration of the site of NRII autophosphorylation to asparagine (H-139-->N [H139N]) or deletion of the C-terminal 59 amino acids of NRII (ter291) resulted in proteins that were not autophosphorylated upon incubation with ATP. Alteration of glycine 313 to alanine resulted in a protein (G313A) that was phosphorylated to a lesser extent than the wild-type protein. Unlike wild-type NRII and H139N, G313A could not be efficiently cross-linked to [alpha-32P]ATP, suggesting that the G313A mutation affects nucleotide binding. Fusion of maltose-binding protein (MBP) to the N-terminal end of NRII resulted in a protein (MBP-NRII) that autophosphorylated normally. We developed a procedure for forming mixed dimers in vitro from these proteins. In mixed dimers consisting of MBP-NRII and H139N, only the MBP-NRII subunit is phosphorylated. In contrast, in mixed dimers consisting of MBP-NRII and G313A, phosphorylation is predominantly on the G313A subunit. We also demonstrated that the G313A and H139N proteins could complement for the autophosphorylation reaction when they were treated so as to permit the formation of mixed dimers and that the wild-type and H139N proteins could phosphorylate the ter291 protein. These results indicate that the autophosphorylation reaction occurs within the dimer by a trans, intersubunit mechanism in which one subunit binds ATP and phosphorylates the other subunit.

Amino Acid Sequence↗