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1.
CtsR, the global heat shock repressor in low GC, Gram+ bacteria, regulates a crucial subset of genes involved in protein quality control. CtsR de-repression occurs not only during heat stress but also during a variety of other environmental stresses, most notably thiol-specific oxidative stress. Here we report that McsA acts as a molecular redox switch that regulates CtsR de-repression via the activation of McsB. Once critical thiols of McsA become oxidized, the strong interaction between McsA and McsB is interrupted and free McsB is no longer inhibited by McsA, resulting in the inactivation of CtsR. This mechanism differs significantly from inactivation of CtsR during heat stress demonstrating a dual activity control of CtsR. Moreover, we show that in those low GC, Gram+ bacteria, which lack the McsA/McsB complex, the Zn finger protein ClpE is able to sense and respond to oxidative stress, also resulting in CtsR inactivation.  相似文献   

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A new kind of prokaryotic protein tyrosine kinase was recently discovered, utilizing a guanidino-phosphotransferase domain for its kinase activity. Guanidino kinase domains originate from eukaryotic phosphagen kinases, a family of phosphoryl transfer enzymes with no homology to the serine/threonine and tyrosine kinase superfamily. Nevertheless, this kinase, McsB, exhibits the main structural and functional properties of prokaryotic tyrosine kinases. Tyrosine phosphorylation in bacteria is predominantly described to be involved in the regulation of exopolysaccharide synthesis and is therefore required for biofilm formation and virulence. McsB on the other hand modulates together with its activator protein, McsA, the activity of the repressor of the class III heat shock genes in B. subtilis. The analogy of the kinase mechanism of McsB to tyrosine kinases implicates that tyrosine kinases may harbor various and independently evolved domains for ATP-binding/hydrolysis and the transfer of the gamma-phosphate of ATP onto tyrosine residues.  相似文献   

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McsA is a key modulator of stress response in Staphylococcus aureus that contains four CXXC potential metal-binding motifs at the N-terminal. Staphylococcus aureus ctsR operon encodes ctsR, clpC, and putative mcsA and mcsB genes. The expression of the ctsR operon in S. aureus was shown to be induced in response to various types of heavy metals such as copper and cadmium. McsA was cloned and overexpressed, and purified product was tested for metal-binding activity. The protein bound to Cu(II), Zn(II), Co(II), and Cd(II). No binding with any heavy metal except copper was found when we performed site-directed mutagenesis of Cys residues of three CXXC motifs of McsA. These data suggest that two conserved cysteine ligands provided by one CXXC motif are required to bind copper ions. In addition, using a bacterial two-hybrid system, McsA was found to be able to bind to McsB and CtsR of S. aureus and the CXXC motif was needed for the binding. This indicates that the Cys residues in the CXXC motif are involved in metal binding and protein interaction.  相似文献   

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Protein quality networks are required for the maintenance of proper protein homeostasis and essential for viability and growth of all living organisms. Hence, regulation and coordination of these networks are critical for survival during stress as well as for virulence of pathogenic species. In low GC, Gram‐positive bacteria central protein quality networks are under the control of the global repressor CtsR. Here, we provide evidence that CtsR activity during heat stress is mediated by intrinsic heat sensing through a glycine‐rich loop, probably in all Gram‐positive species. Moreover, a function for the recently identified arginine kinase McsB is confirmed, however, not for initial inactivation and dissociation of CtsR from the DNA, but for heat‐dependent auto‐activation of McsB as an adaptor for ClpCP‐mediated degradation of CtsR.  相似文献   

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The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level. In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of clpP encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE. Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression. Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpP-dependent processing and that disruption of the zinc finger domain renders ClpE more susceptible. Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in Bacillus subtilis. Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR.  相似文献   

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Controlled protein degradation is an important cellular reaction for the fast and efficient adaptation of bacteria to ever-changing environmental conditions. In the low-GC, Gram-positive model organism Bacillus subtilis, the AAA+ protein ClpC requires specific adaptor proteins not only for substrate recognition but also for chaperone activity. The McsB adaptor is activated particularly during heat stress, allowing the controlled degradation of the CtsR repressor by the ClpCP protease. Here we report how the McsB adaptor becomes activated by autophosphorylation on specific arginine residues during heat stress. In nonstressed cells McsB activity is inhibited by ClpC as well as YwlE.  相似文献   

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During the development of transformability (competence), Bacillus subtilis synthesizes a set of proteins that mediate both the uptake of DNA at the cell poles and the recombination of this DNA with the resident chromosome. Most, if not all, of these Com proteins localize to the poles of the cell, where they associate with one another, and are then seen to delocalize as transformability declines. In this study, we use fluorescence microscopy to analyse the localization and delocalization processes. We show that localization most likely occurs by a diffusion-capture mechanism, not requiring metabolic energy, whereas delocalization is prevented in the presence of sodium azide. The kinetics of localization suggest that this process requires the synthesis of a critical protein or set of proteins, which are needed to anchor the Com protein complex to the poles. We further show that the protein kinase proteins McsA and McsB are needed for delocalization, as are ClpP and either of the AAA+ ( A TPases a ssociated with a variety of cellular a ctivities) proteins ClpC or ClpE. Of these proteins, at least McsB, ClpC and ClpP localize to the cell poles of competent cells. Our evidence strongly suggests that delocalization depends on the degradation of the postulated anchor protein(s) by the McsA-McsB-(ClpC or ClpE)-ClpP protease in an ATP-dependent process that involves the autophosphorylation of McsB. The extent of cell–pole association at any given time reflects the relative rates of localization and delocalization. The kinetics of this dynamic process differs for individual Com proteins, with the DNA-binding proteins SsbB and DprA exhibiting less net localization.  相似文献   

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The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knockout strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.  相似文献   

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We have recently reported that fluoride interacts directly with the insulin receptor, which causes inhibition of its phosphotransferase activity. The inhibitory effect of fluoride on phosphotransferase activity is not due to the formation of complexes with aluminium and occurs in the absence of alterations to the binding of ATP or insulin. In this report we substantiate that the tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle shows a strict requirement of Mg2+ ions (Ka near 11 mM). This effect of Mg2+ was inhibited in a competitive manner by Mn2+, which is compatible with competition of both divalent ions for binding sites. The inhibition of tyrosine kinase activity caused by fluoride was dependent on the concentration of Mg2+ in the medium and no inhibitory effect was detected at low concentrations of Mg2+. Moreover, the addition of increasing concentrations of Mn2+ in the presence of a constant high concentr rease in the inhibitory effect of fluoride. These results indicate that the Mg-insulin receptor complex is the major fluoride-susceptible form. Based on the characteristics of the inhibition of tyrosine kinase shown by fluoride it might be proposed that its action is exerted by the formation of multi-ionic MgF complexes analogous to Pi, which bind to the insulin receptor kinase.  相似文献   

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Creatine kinase (CK) is part of a conserved family of ATP:guanidino phosphotransferases whose members play important roles in intracellular energy flow. Previously characterized members of this family are approximately 80-kDa dimers of two related 40-kDa subunits. We have cloned a gene from the parasitic trematode Schistosoma mansoni which has substantial amino acid sequence similarities to CK. Like the genes for vertebrate CKs, this gene is developmentally regulated; mRNA levels are high in the infective cercarial stage but rapidly decrease upon transformation to the parasitic schistosomulum stage. In contrast to members of the guanidino phosphotransferase family characterized previously, however, the schistosome gene appears to be a direct fusion of two CK-like domains that encode a single 74-kDa polypeptide. Correlative evidence from enzyme assays of crude parasite homogenates suggests that the cloned gene is a creatine kinase. This represents the first molecular cloning of an invertebrate ATP:guanidino phosphotransferase.  相似文献   

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The rat neu oncogene product is a member of the epidermal growth factor (EGF) receptor subgroup of the superfamily of growth factor receptor tyrosine kinases. The oncogenic activation of the neu protein occurs by a point mutation within its transmembrane region which results in an increase in its tyrosine kinase activity. Using three different forms of neu expressed in insect cells via baculovirus infection, we have examined the biochemical differences between the normal and transforming forms of neu and investigated the role of the transmembrane domain in its tyrosine kinase activity. One form of neu which was expressed in insect cells consisted of the complete tyrosine kinase domain but lacked the extracellular and transmembrane regions (designated NTK). The other two forms consisted of the tyrosine kinase domain, the transmembrane domain, and 40 amino acids of the extracellular domain. One of these transmembrane forms of neu contained the normal valine residue at position 664 within the transmembrane region (MS-N), while the other contained the oncogenic glutamic acid residue at this position (MS-T). Direct comparisons of NTK, MS-N, and MS-T have shown that the NTK protein is capable of the highest extents of both autophosphorylation activity and the tyrosine phosphorylation of exogenous substrate, suggesting that the presence of the transmembrane region of neu suppresses the tyrosine kinase activity of this receptor. In addition, we have found that the oncogenic point mutation within the transmembrane region stimulates the tyrosine kinase activity of the neu protein by allowing it to more effectively utilize Mg2+. Overall, the results of these studies suggest that the valine to glutamic acid substitution at position 664 may at least partially relieve a negative constraint imparted by the membrane-spanning domain on the tyrosine kinase activity of neu and enables a more effective use of Mg2+ in the catalysis of tyrosine phosphorylation of exogenous substrates.  相似文献   

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Evidence for two catalytically active kinase domains in pp90rsk.   总被引:12,自引:2,他引:10       下载免费PDF全文
Mitogen-activated protein kinase and one of its targets, pp90rsk (ribosomal S6 kinase [RSK]), represent two serine/threonine kinases in the Ras-activated signalling cascade that are capable of directly regulating gene expression. pp90rsk has been shown to have two highly conserved and distinct catalytic domains. However, whether both domains are active and which domain is responsible for its various identified phosphotransferase activities have not been determined. Here we demonstrate that the N-terminal domain is responsible for its phosphotransferase activity towards a variety of substrates which contain an RXXS motif at the site of in vitro phosphorylation, including serum response factor, c-Fos, Nur77, and the 40S ribosomal protein S6. We also provide evidence that the C-terminal domain is catalytically active and can be further activated by mitogen-activated protein kinase phosphorylation.  相似文献   

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Biological responses to epidermal growth factor (EGF) depend on the ligand-stimulated protein tyrosine kinase activity of its receptor. To further characterize the enzymatic activity of the EGF receptor, the baculovirus expression system was used to express the cytoplasmic protein tyrosine kinase domain of the EGF receptor. Spodoptera frugiperda (Sf9) cells infected with recombinant baculovirus correctly expressed an active tyrosine kinase domain of the EGF receptor as demonstrated by 35S metabolic labeling, immunoblotting with anti-EGF receptor and anti-phosphotyrosine antibodies, and autophosphorylation analysis. The kinase domain (Mr 66,000) was purified to near homogeneity using a monoclonal anti-phosphotyrosine antibody column, providing 0.5 mg of kinase domain/liter of Sf9 cells (23% yield). The purified kinase domain exhibited a strong preference for Mn2+ compared to Mg2+. The specific activity of the kinase domain was low compared to purified, EGF-activated EGF receptor. However, the addition of sphingosine or ammonium sulfate greatly increased the activity of the kinase domain to equal or exceed the activity of ligand-activated holo EGF receptor. These results indicate that the addition of sphingosine or ammonium sulfate to the purified kinase domain can mimic the effect of EGF to induce a conformation of the holo EGF receptor which is optimal for tyrosine kinase activity. Deletion of the ligand binding domain, analogous to that which occurs in erb B, is not sufficient to fully activate the kinase, implying that EGF causes conformational changes additional to removal of an inhibitory constraint.  相似文献   

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