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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Histidine-containing protein (HPr) of gram-positive bacteria was found to be phosphorylated at a seryl residue (P-ser-HPr) in an ATP-dependent reaction catalyzed by a protein kinase (J. Deutscher and M. H. Saier, Jr., Proc. Natl. Acad. Sci. U.S.A. 80:6790-6794, 1983). Here we describe the purification and characterization of a soluble enzyme of Streptococcus faecalis which splits the phosphoryl bond in P-ser-HPr. The enzyme has a molecular weight of ca. 7.5 X 10(4), as determined by its migration behavior on a Sephacryl S-200 column. On native polyacrylamide gels the purified enzyme produced only one protein band. On sodium dodecyl sulfate-polyacrylamide gels we found one major protein band of molecular weight 2.9 X 10(4) and two minor protein bands of molecular weights 2.3 X 10(4) and 7 X 10(4). Fructose 1,6-diphosphate, which stimulated the ATP-dependent, protein kinase-catalyzed phosphorylation of HPr, had no effect on the phosphatase activity. Other glycolytic intermediates also had no effect. However, inorganic phosphate, which inhibited the ATP-dependent HPr kinase, stimulated the P-ser-HPr phosphatase. EDTA at a concentration of 0.1 mM completely inhibited the phosphatase. Divalent cations like Mg2+, Mn2+, and Co2+ overcame the inhibition by EDTA. Fe2+, Zn2+, and Cu2+ had no effect, whereas Ca2+ slightly inhibited the phosphatase. ATP was also found to inhibit the phosphatase. Under conditions in which ATP severely inhibited the phosphatase, ADP was found to have no effect on the enzyme activity. Besides P-ser-HPr of S. faecalis, the phosphatase was also able to hydrolyze the phosphoryl bond in P-ser-HPr of Streptococcus lactis, Staphylococcus aureus, Bacillus subtilis, Streptococcus pyogenes, and Lactobacillus casei. Phosphoenolpyruvate-dependent o-nitrophenyl-beta-D-galactopyranoside phosphorylation, catalyzed by the S. aureus phosphoenolpyruvate:lactose phosphotransferase system, was about 150-fold decreased in the presence of P-ser-HPr of S. aureus, as compared with HPr. However, when P-ser-HPr was first incubated with P-ser-HPr phosphatase to allow complete hydrolysis of the phosphoryl bond, it had the same activity as HPr. Besides this cytoplasmic phosphoprotein phosphatase, we detected a membrane-bound phosphatase which also hydrolyzed the phosphoryl bond in P-ser-HPr.
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PMID:Streptococcal phosphoenolpyruvate: sugar phosphotransferase system: purification and characterization of a phosphoprotein phosphatase which hydrolyzes the phosphoryl bond in seryl-phosphorylated histidine-containing protein. 299 39

Streptococcus bovis had a diauxic pattern of glucose and lactose utilization, and both of these sugars were transported by the sugar phosphotransferase system (PTS). Lactose catabolism was inducible, and S. bovis used the tagatose pathway to ferment lactose. Since a mutant that was deficient in glucose PTS activity transported lactose as fast as the wild-type, it appeared that S. bovis has separate enzyme IIs for glucose and lactose. The nonmetabolizable glucose analogue 2-deoxyglucose (2-DG) was a noncompetitive inhibitor of methyl beta-D-thiogalactopyranoside (TMG) transport, and cells that were provided with either glucose or 2-DG were unable to transport TMG or lactose. Because the glucose-PTS-deficient mutant could ferment glucose, but could not exclude TMG, it appeared that enzyme IIGlc rather than glucose catabolism per se was the critical feature of inducer exclusion. Cells that had accumulated TMG as TMG 6-phosphate expelled free TMG when glucose was added, but 2-DG was unable to cause TMG expulsion. The glucose-PTS-deficient mutant could still expel TMG in the presence of exogenous glucose. Membrane vesicles also exhibited glucose-dependent TMG exclusion and TMG expulsion. Membrane vesicles that were electroporated with phosphoenolpyruvate (PEP) and HPr retained TMG for more than 3 min, but vesicles that were electroporated with PEP plus HPr and fructose 1,6-diphosphate (FDP) (or glycerate 2-phosphate) lost their ability to retain TMG. Because FDP was able to trigger the ATP-dependent phosphorylation of HPr, it appeared that inducer expulsion was mediated by an FDP-activated protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of the lactose phosphotransferase system of Streptococcus bovis by glucose: independence of inducer exclusion and expulsion mechanisms. 749 38

Lactobacillus brevis accumulates lactose and nonmetabolizable lactose analogues via sugar/H+ symport, but addition of glucose to the extracellular medium results in rapid efflux of the free sugar from the cells due to the uncoupling of sugar transport from proton transport. By using vesicles of L. brevis cells, we recently showed that these regulatory/effects could be attributed to the metabolite-activated ATP-dependent protein kinase-catalyzed phosphorylation of serine-46 in the phosphocarrier protein HPr [HPr(Ser-P)] of the phosphotransferase system and that a mutant form of HPr with the serine-46-->aspartate replacement ([S46D]HPr) is apparently locked in the seryl phosphorylated conformation. We here demonstrate that [S46D]HPr binds directly to inside-out membrane vesicles of L. brevis that contain the lactose permease. Sugar substrates of the permease markedly and specifically stimulate binding of [S46D]HPr to the membranes while certain transport inhibitors such as N-ethylmaleimide block binding. The pH dependency for binding follows that for transport. Wild-type HPr and the [S46A]HPr mutant protein did not appreciably compete with [S46D]HPr for binding to the permease. These results provide evidence for the direct interaction of HPr(Ser-P) with an allosteric site on the lactose/proton symporter of L. brevis for the purpose of regulating sugar accumulation in response to the metabolic needs of the cell.
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PMID:Cooperative binding of lactose and the phosphorylated phosphocarrier protein HPr(Ser-P) to the lactose/H+ symport permease of Lactobacillus brevis. 783 2

Lactobacillus brevis takes up glucose and the nonmetabolizable glucose analog 2-deoxyglucose (2DG), as well as lactose and the nonmetabolizable lactose analoge thiomethyl beta-galactoside (TMG), via proton symport. Our earlier studies showed that TMG, previously accumulated in L. brevis cells via the lactose:H+ symporter, rapidly effluxes from L. brevis cells or vesicles upon addition of glucose and that glucose inhibits further accumulation of TMG. This regulation was shown to be mediated by a metabolite-activated protein kinase that phosphorylase serine 46 in the HPr protein. We have now analyzed the regulation of 2DG uptake and efflux and compared it with that of TMG. Uptake of 2DG was dependent on an energy source, effectively provided by intravesicular ATP or by extravesicular arginine which provides ATP via an ATP-generating system involving the arginine deiminase pathway. 2DG uptake into these vesicles was not inhibited, and preaccumulated 2DG did not efflux from them upon electroporation of fructose 1,6-diphosphate or gluconate 6-phosphate into the vesicles. Intravesicular but not extravesicular wild-type or H15A mutant HPr of Bacillus subtilis promoted inhibition (53 and 46%, respectively) of the permease in the presence of these metabolites. Counterflow experiments indicated that inhibition of 2DG uptake is due to the partial uncoupling of proton symport from sugar transport. Intravesicular S46A mutant HPr could not promote regulation of glucose permease activity when electroporated into the vesicles with or without the phosphorylated metabolites, but the S46D mutant protein promoted regulation, even in the absence of a metabolite. The Vmax but not the Km values for both TMG and 2DG uptake were affected. Uptake of the natural, metabolizable substrates of the lactose, glucose, mannose, and ribose permeases was inhibited by wild-type HPr in the presence of fructose 1,6-diphosphate or by S46D mutant HPr. These results establish that HPr serine phosphorylation by the ATP-dependent, metabolite-activated HPr kinase regulates glucose and lactose permease activities in L. brevis and suggest that other permeases may also be subject to this mode of regulation.
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PMID:Regulation of the glucose:H+ symporter by metabolite-activated ATP-dependent phosphorylation of HPr in Lactobacillus brevis. 820 25

L-29, a mammalian soluble lactose-binding lectin, was previously shown to be phosphorylated in confluent 3T3 fibroblasts (Cowles, E. A., Agrwal, N., Anderson, R. L., and Wang, J. L. (1990) J. Biol. Chem. 265, 17706-17712), which contain a small amount of this protein. We have determined the site of phosphorylation on L-29, taking advantage of the abundance of L-29 (about 1% of total soluble cell protein) in confluent polarized Madin-Darby canine kidney (MDCK) cells. Approximately 15-20% of the L-29 is phosphorylated in these cells. Phosphoamino acid analysis showed phosphate incorporation only at serine. Analysis of chymotryptic and endoproteinase Asp-N-generated NH2-terminal fragments by Edman degradation showed that 90% of the phosphate was at Ser6 and 10% at Ser12. The sequence surrounding Ser6, which is conserved in all known L-29 sequences, indicated that this serine might be phosphorylated by casein kinase I or casein kinase II. Reaction of human recombinant L-29 with [gamma-32P]ATP and each of these casein kinases showed that only casein kinase I catalyzed significant incorporation of 32P into L-29; and, as with the L-29 from the MDCK cell extracts, most of the phosphate was incorporated at Ser6 and a small amount was incorporated at Ser12.
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PMID:L-29, a soluble lactose-binding lectin, is phosphorylated on serine 6 and serine 12 in vivo and by casein kinase I. 825 6

Results currently available clearly indicate that the metabolite-activated protein kinase-mediated phosphorylation of Ser-46 in HPr plays a key role in catabolite repression and the control of inducer levels in low-GC Gram-positive bacteria. This protein kinase is not found in enteric bacteria such as E. coli and Salmonella typhimurium where an entirely different PTS-mediated regulatory mechanism is responsible for catabolite repression and inducer concentration control. In Table 2 these two mechanistically dissimilar but functionally related processes are compared (Saier et al., 1995b). In Gram-negative enteric bacteria, an external sugar is sensed by the sugar-recognition constituent of an Enzyme II complex of the PTS (IIC), and a dephosphorylating signal is transmitted via the Enzyme IIB/HPr proteins to the central regulatory protein, IIAGlc. Targets regulated include (1) permeases specific for lactose, maltose, melibiose and raffinose, (2) catabolic enzymes such as glycerol kinase that generate cytoplasmic inducers, and (3) the cAMP biosynthetic enzyme, adenylate cyclase that mediates catabolite repression (Saier, 1989, 1993). In low-GC Gram-positive bacteria, cytoplasmic phosphorylated sugar metabolites are sensed by the HPr kinase which is allostericlaly activated. HPr becomes phosphorylated on Ser-46, and this phosphorylated derivative regulates the activities of its target proteins. These targets include (1) the PTS, (2) non-PTS permeases (both of which are inhibited) and (3) a cytoplasmic sugar-P phosphatase which is activated to reduce cytoplasmic inducer levels. Other important targets of HPr(ser-P) action are (4) the CcpA protein and probably (5) the CepB transcription factor. These two proteins together are believed to determine the intensity of catabolite repression. Their relative importance depends on physiological conditions. Both proteins may respond to the cytoplasmic concentration of HPr(ser-P) and appropriate metabolites. CepA possibly binds sugar metabolites such as FBP as well as HPr(ser-P). Because HPr(his-P, ser-P) does not bind to CepA, the regulatory cascade is also sensitive to the external PTS sugar concentration. Mutational analyses (unpublished results) suggest that CepA may bind to a site that includes His-15. Interestingly, both the CepA protein in the Gram-positive bacterium, B. subtilis, and glycerol kinase in the Gram-negative bacterium, E. coli, sense both a PTS protein and a cytoplasmic metabolic intermediate. The same may be true of target permeases and enzymes in both types of organisms, but this possibility has not yet been tested. The parallels between the Gram-negative and Gram-positive bacterial regulatory systems are superficial at the mechanistic level but fundamental at the functional level. Thus, the PTS participates in regulation in both cases, and phosphorylation of its protein constituents plays key roles. However, the stimuli sensed, the transmission mechanisms, the central PTS regulatory proteins that effect allosteric regulation, and some of the target proteins are completely different. It seems clear that these two transmission mechanisms evolved independently. They provide a prime example of functional convergence.
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PMID:Catabolite repression and inducer control in Gram-positive bacteria. 893 96

Phosphorylation of HPr on a serine residue at position 46 (Ser-46) by an ATP-dependent protein kinase has been reported in several Gram-positive bacteria, and the resulting intermediate, HPr(Ser-P), has been shown to mediate inducer exclusion in lactococci and lactobacilli and catabolite repression in Bacillus subtilis and Bacillus megaterium. We report here the phenotypic properties of an isogenic spontaneous mutant (G22.4) of Streptococcus salivarius ATCC 25975, in which a missense mutation results in the replacement of isoleucine at position 47 (Ile-47) by threonine (Thr) in HPr. This substitution did not prevent the phosphorylation of HPr on Ser-46, nor did it impede the phosphorylation of HPr on His-15 by EI or the transfer of the phosphoryl group from HPr(His-P) to other PTS proteins. However, the 147T substitution did perturb, in glucose-grown but not in galactose-grown cells, the cellular equilibrium between the various forms of HPr, resulting in an increase in the amount of free HPr at the expense of HPr(His-P)(Ser-P); the levels of HPr(His-P) and HPr(Ser-P) were not affected. Growth on melibiose was virtually identical for the wild-type and mutant strains, whereas the generation time of the mutant on the other sugars tested (glucose, fructose, mannose, lactose and galactose) increased 1.2- to 1.5-fold. The preferential metabolism of PTS sugars (glucose and fructose) over non-PTS sugars (lactose and melibiose) that is observed in wild-type cells was abolished in cells of mutant G22.4. Moreover, alpha- and beta-galactosidases were derepressed in glucose- and fructose-grown cells of the mutant. The data suggest that HPr regulates the preferential metabolism of PTS sugars over the non-PTS sugars, lactose and melibiose, through the repression of the pertinent catabolic genes. This HPr-dependent repression, however, seems to occur solely when cells are growing on a PTS sugar.
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PMID:Replacement of isoleucine-47 by threonine in the HPr protein of Streptococcus salivarius abrogates the preferential metabolism of glucose and fructose over lactose and melibiose but does not prevent the phosphorylation of HPr on serine-46. 937 99

In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His(15)) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser(46)) by an ATP-dependent protein kinase (His approximately P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His(15) by EI nor the phosphorylation at Ser(46) by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His approximately P) than the wild-type strain, while the levels of free HPr and HPr(His approximately P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of alpha-galactosidase, beta-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.
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PMID:Phenotypic consequences resulting from a methionine-to-valine substitution at position 48 in the HPr protein of Streptococcus salivarius. 1055 56

Bacillus subtilis can utilize several sugars as single sources of carbon and energy. Many of these sugars are transported and concomitantly phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system (PTS). In addition to its role in sugar uptake, the PTS is one of the major signal transduction systems in B. subtilis. In this study, an analysis of the complete set of PTS proteins encoded within the B. subtilis genome is presented. Fifteen sugar-specific PTS permeases were found to be present and the functions of novel PTS permeases were studied based on homology to previously characterized permeases, analysis of the structure of the gene clusters in which the permease encoding genes are located and biochemical analysis of relevant mutants. Members of the glucose, sucrose, lactose, mannose and fructose/mannitol families of PTS permeases were identified. Interestingly, nine pairs of IIB and IIC domains belonging to the glucose and sucrose permease families are present in B. subtilis; by contrast only five Enzyme IIA(Glc)-like proteins or domains are encoded within the B. subtilis genome. Consequently, some of the EIIA(Glc)-like proteins must function in phosphoryl transfer to more than one IIB domain of the glucose and sucrose families. In addition, 13 PTS-associated proteins are encoded within the B. subtilis genome. These proteins include metabolic enzymes, a bifunctional protein kinase/phosphatase, a transcriptional cofactor and transcriptional regulators that are involved in PTS-dependent signal transduction. The PTS proteins and the auxiliary PTS proteins represent a highly integrated network that catalyses and simultaneously modulates carbohydrate utilization in this bacterium.
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PMID:Novel phosphotransferase system genes revealed by genome analysis - the complete complement of PTS proteins encoded within the genome of Bacillus subtilis. 1062 40

Ecalectin/galectin-9 was recently described as a novel eosinophil chemoattractant highly expressed in immune tissues. We investigated the regulation of galectin-9 expression and release in Jurkat (a T cell line) cells. We demonstrated that medium and long-sized galectin-9 isoforms were constitutively expressed, and phorbol 12-myriastate 13-acetate (PMA) upregulated the level of galectin-9 mRNA in Jurkat cells. Western blotting and flow cytometry analyses revealed that PMA stimulation resulted in the upregulation of both intracellular and surface galectin-9 protein. The stimulated Jurkat cells simultaneously released evident eosinophil chemoattractant activity (ECA). Main ECA was adsorbed by both lactose and anti-galectin-9 antibody affinity column, suggesting that the ECA was ascribed to galectin-9. When Jurkat cells were stimulated with PMA in the presence of a BB94, a matrix metalloproteinase (MMP) inhibitor, but not tissue inhibitor of metalloproteinase-1 (TIMP-1), the release of galectin-9 was suppressed in a dose-dependent manner. We further found that calphostin c, a protein kinase c (PKC) inhibitor, weakly but significantly suppressed the release of galectin-9. The present data suggested that galectin-9 production in Jurkat cells is provoked by the stimulation with PMA and that some MMP and PKC is, at least, partly involved in the release of galectin-9 from Jurkat cells.
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PMID:Regulation of galectin-9 expression and release in Jurkat T cell line cells. 1188 44


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