scholarly journals Cysteine-Scanning Analysis of the Dimerization Domain of EnvZ, an Osmosensing Histidine Kinase

2003 ◽  
Vol 185 (11) ◽  
pp. 3429-3435 ◽  
Author(s):  
Ling Qin ◽  
Shengjian Cai ◽  
Yan Zhu ◽  
Masayori Inouye
Keyword(s):  
Phosphatase Activity ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Dimerization Domain ◽  
Phosphatase Domain ◽  
Mutant Proteins ◽  
Cysteine Scanning ◽  
Substitution Mutations ◽  
Cognate Response Regulator

ABSTRACT EnvZ and OmpR are a transmembrane sensor and its cognate response regulator, respectively, regulating the transcription of porin genes in response to medium osmolarity in Escherichia coli. The cytoplasmic domain of EnvZ (EnvZc) possesses both kinase and phosphatase activities and can be dissected into two functional domains, A and B. Here, we performed a cysteine-scanning analysis of domain A, a 67-residue central dimerization and phosphatase domain containing His-243 as the phosphorylation site, and we examined the effects of the cysteine substitution mutations on the enzymatic activities of domain A. The substitution mutations were made at 31 residues, from which 24 mutant domain A proteins were biochemically characterized. From the analysis of the phosphatase activity of purified mutant proteins, it was found that there are two regions in domain A which are important for this activity. Cysteine mutations in these regions dramatically reduce or completely abolish the phosphatase activity of domain A. The mutations that have the most-severe effects on domain A phosphatase activity also significantly reduce the phosphatase activity of EnvZc containing the same mutation. Using an in vitro complementation system with EnvZc(H243V), these cysteine mutants were further characterized for their autophosphorylation activities as well as their phosphotransfer activities. The results indicate that some mutations are specific either for the phosphatase activity or for the kinase activity.

scholarly journals Requirement of PKR Dimerization Mediated by Specific Hydrophobic Residues for Its Activation by Double-Stranded RNA and Its Antigrowth Effects in Yeast

1998 ◽  
Vol 18 (12) ◽  
pp. 7009-7019 ◽  
Author(s):  
Rekha C. Patel ◽  
Ganes C. Sen
Keyword(s):  
Helical Structure ◽  
Wild Type ◽  
Dimerization Domain ◽  
Double Stranded Rna ◽  
Mutant Proteins ◽  
Hydrophobic Residues ◽  
Dsrna Binding ◽  
Dependent Protein ◽  
Substitution Mutations

ABSTRACT The roles of protein dimerization and double-stranded RNA (dsRNA) binding in the biochemical and cellular activities of PKR, the dsRNA-dependent protein kinase, were investigated. We have previously shown that both properties of the protein are mediated by the same domain. Here we show that dimerization is mediated by hydrophobic residues present on one side of an amphipathic α-helical structure within this domain. Appropriate substitution mutations of residues on that side produced mutants with increased or decreased dimerization activities. Using these mutants, we demonstrated that dimerization is not essential for dsRNA binding. However, enhancing dimerization artificially, by providing an extraneous dimerization domain, increased dsRNA binding of both wild-type and mutant proteins. In vitro, the dimerization-defective mutants could not be activated by dsRNA but were activated normally by heparin. In Saccharomyces cerevisiae, unlike wild-type PKR, these mutants could not inhibit cell growth and the dsRNA-binding domain of the dimerization-defective mutants could not prevent the antigrowth effect of wild-type PKR. These results demonstrate the biological importance of the dimerization properties of PKR.

scholarly journals Quantitative Kinetic Analyses of Shutting Off a Two-Component System

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Rong Gao ◽  
Ann M. Stock
Keyword(s):  
Phosphatase Activity ◽  
Response Regulator ◽  
Two Component System ◽  
Component System ◽  
Signaling Systems ◽  
Cellular Environment ◽  
Two Component ◽  
The Impact

ABSTRACT Cells rely on accurate control of signaling systems to adapt to environmental perturbations. System deactivation upon stimulus removal is as important as activation of signaling pathways. The two-component system (TCS) is one of the major bacterial signaling schemes. In many TCSs, phosphatase activity of the histidine kinase (HK) is believed to play an essential role in shutting off the pathway and resetting the system to the prestimulus state. Two basic challenges are to understand the dynamic behavior of system deactivation and to quantitatively evaluate the role of phosphatase activity under natural cellular conditions. Here we report a kinetic analysis of the response to shutting off the archetype Escherichia coli PhoR-PhoB TCS pathway using both transcription reporter assays and in vivo phosphorylation analyses. Upon removal of the stimulus, the pathway is shut off by rapid dephosphorylation of the PhoB response regulator (RR) while PhoB-regulated gene products gradually reset to prestimulus levels through growth dilution. We developed an approach combining experimentation and modeling to assess in vivo kinetic parameters of the phosphatase activity with kinetic data from multiple phosphatase-diminished mutants. This enabled an estimation of the PhoR phosphatase activity in vivo , which is much stronger than the phosphatase activity of PhoR cytoplasmic domains analyzed in vitro . We quantitatively modeled how strong the phosphatase activity needs to be to suppress nonspecific phosphorylation in TCSs and discovered that strong phosphatase activity of PhoR is required for cross-phosphorylation suppression. IMPORTANCE Activation of TCSs has been extensively studied; however, the kinetics of shutting off TCS pathways is not well characterized. We present comprehensive analyses of the shutoff response for the PhoR-PhoB system that reveal the impact of phosphatase activity on shutoff kinetics. This allows development of a quantitative framework not only to characterize the phosphatase activity in the natural cellular environment but also to understand the requirement for specific strengths of phosphatase activity to suppress nonspecific phosphorylation. Our model suggests that the ratio of the phosphatase rate to the nonspecific phosphorylation rate correlates with TCS expression levels and the ratio of the RR to HK, which may contribute to the great diversity of enzyme levels and activities observed in different TCSs.

scholarly journals Phosphorylation of the Pseudomonas aeruginosa Response Regulator AlgR Is Essential for Type IV Fimbria-Mediated Twitching Motility

2002 ◽  
Vol 184 (16) ◽  
pp. 4544-4554 ◽  
Author(s):  
Cynthia B. Whitchurch ◽  
Tatiana E. Erova ◽  
Jacqui A. Emery ◽  
Jennifer L. Sargent ◽  
Jonathan M. Harris ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Twitching Motility ◽  
Type Iv ◽  
Molecular Events ◽  
And Function ◽  
Alginate Biosynthesis

ABSTRACT The response regulator AlgR is required for both alginate biosynthesis and type IV fimbria-mediated twitching motility in Pseudomonas aeruginosa. In this study, the roles of AlgR signal transduction and phosphorylation in twitching motility and biofilm formation were examined. The predicted phosphorylation site of AlgR (aspartate 54) and a second aspartate (aspartate 85) in the receiver domain of AlgR were mutated to asparagine, and mutant algR alleles were introduced into the chromosome of P. aeruginosa strains PAK and PAO1. Assays of these mutants demonstrated that aspartate 54 but not aspartate 85 of AlgR is required for twitching motility and biofilm initiation. However, strains expressing AlgR D85N were found to be hyperfimbriate, indicating that both aspartate 54 and aspartate 85 are involved in fimbrial biogenesis and function. algD mutants were observed to have wild-type twitching motility, indicating that AlgR control of twitching motility is not mediated via its role in the control of alginate biosynthesis. In vitro phosphorylation assays showed that AlgR D54N is not phosphorylated by the enteric histidine kinase CheA. These findings indicate that phosphorylation of AlgR most likely occurs at aspartate 54 and that aspartate 54 and aspartate 85 of AlgR are required for the control of the molecular events governing fimbrial biogenesis, twitching motility, and biofilm formation in P. aeruginosa.

scholarly journals Proposed Signal Transduction Role for Conserved CheY Residue Thr87, a Member of the Response Regulator Active-Site Quintet

1998 ◽  
Vol 180 (14) ◽  
pp. 3563-3569 ◽  
Author(s):  
Jeryl L. Appleby ◽  
Robert B. Bourret
Keyword(s):  
Amino Acid ◽  
Active Site ◽  
Response Regulator ◽  
Hydroxyl Group ◽  
Amino Acid Substitutions ◽  
Mutant Proteins ◽  
Hydroxy Amino Acid ◽  
Hydroxy Amino ◽  
Flagellar Rotation

ABSTRACT CheY serves as a structural prototype for the response regulator proteins of two-component regulatory systems. Functional roles have previously been defined for four of the five highly conserved residues that form the response regulator active site, the exception being the hydroxy amino acid which corresponds to Thr87 in CheY. To investigate the contribution of Thr87 to signaling, we characterized, genetically and biochemically, several cheY mutants with amino acid substitutions at this position. The hydroxyl group appears to be necessary for effective chemotaxis, as a Thr→Ser substitution was the only one of six tested which retained a Che+ swarm phenotype. Although nonchemotactic, cheY mutants with amino acid substitutions T87A and T87C could generate clockwise flagellar rotation either in the absence of CheZ, a protein that stimulates dephosphorylation of CheY, or when paired with a second site-activating mutation, Asp13→Lys, demonstrating that a hydroxy amino acid at position 87 is not essential for activation of the flagellar switch. All purified mutant proteins examined phosphorylated efficiently from the CheA kinase in vitro but were impaired in autodephosphorylation. Thus, the mutant CheY proteins are phosphorylated to a greater degree than wild-type CheY yet support less clockwise flagellar rotation. The data imply that Thr87 is important for generating and/or stabilizing the phosphorylation-induced conformational change in CheY. Furthermore, the various position 87 substitutions differentially affected several properties of the mutant proteins. The chemotaxis and autodephosphorylation defects were tightly linked, suggesting common structural elements, whereas the effects on self-catalyzed and CheZ-mediated dephosphorylation of CheY were uncorrelated, suggesting different structural requirements for the two dephosphorylation reactions.

scholarly journals The Arabidopsis Rho of Plants GTPase ROP1 Is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate

Plants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2053
Author(s):  
Dalma Ménesi ◽  
Éva Klement ◽  
Györgyi Ferenc ◽  
Attila Fehér
Keyword(s):  
Protein Kinases ◽  
Phosphorylation Site ◽  
Interaction Strength ◽  
Yeast Cells ◽  
Phosphorylation Sites ◽  
Mutant Proteins ◽  
In Vivo Test ◽  
Calcium Dependent

Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test.

scholarly journals Identification of Novel Thermosensors in Gram-Positive Pathogens

2020 ◽  
Vol 7 ◽  
Author(s):  
Pilar Fernández ◽  
Alejandra Raquel Díaz ◽  
María Florencia Ré ◽  
Lucía Porrini ◽  
Diego de Mendoza ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Response Regulator ◽  
Physiological Mechanism ◽  
Virulence Gene ◽  
Membrane Lipid ◽  
Gram Positive ◽  
Virulence Gene Expression ◽  
Cognate Response Regulator

Temperature is a crucial variable that every living organism, from bacteria to humans, need to sense and respond to in order to adapt and survive. In particular, pathogenic bacteria exploit host-temperature sensing as a cue for triggering virulence gene expression. Here, we have identified and characterized two integral membrane thermosensor histidine kinases (HKs) from Gram-positive pathogens that exhibit high similarity to DesK, the extensively characterized cold sensor histidine kinase from Bacillus subtilis. Through in vivo experiments, we demonstrate that SA1313 from Staphylococcus aureus and BA5598 from Bacillus anthracis, which likely control the expression of putative ATP binding cassette (ABC) transporters, are regulated by environmental temperature. We show here that these HKs can phosphorylate the non-cognate response regulator DesR, partner of DesK, both in vitro and in vivo, inducing in B. subtilis the expression of the des gene upon a cold shock. In addition, we report the characterization of another DesK homolog from B. subtilis, YvfT, also closely associated to an ABC transporter. Although YvfT phosphorylates DesR in vitro, this sensor kinase can only induce des expression in B. subtilis when overexpressed together with its cognate response regulator YvfU. This finding evidences a physiological mechanism to avoid cross talk with DesK after a temperature downshift. Finally, we present data suggesting that the HKs studied in this work appear to monitor different ranges of membrane lipid properties variations to mount adaptive responses upon cooling. Overall, our findings point out that bacteria have evolved sophisticated mechanisms to assure specificity in the response to environmental stimuli. These findings pave the way to understand thermosensing mediated by membrane proteins that could have important roles upon host invasion by bacterial pathogens.

scholarly journals The CckA-ChpT-CtrA phosphorelay controlling Rhodobacter capsulatus gene transfer agent (RcGTA) production is bi-directional and regulated by cyclic-di-GMP

2020 ◽  
Author(s):  
Reynold G. Farrera-Calderon ◽  
Purvikalyan Pallegar ◽  
Alexander B. Westbye ◽  
Christina Wiesmann ◽  
Andrew S. Lang ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Phosphatase Activity ◽  
Regulatory Networks ◽  
Rhodobacter Capsulatus ◽  
Response Regulator ◽  
Evolutionary Significance ◽  
Secondary Messenger ◽  
Gene Transfer Agent

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, histidine phosphotransferase ChpT and response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA to biochemically demonstrate that they form a phosphorelay. The secondary messenger cyclic-di-GMP changed CckA from a kinase to a phosphatase resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro, and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for lytic release of RcGTA. The binding of cyclic-di-GMP to the wild type and mutant CckA proteins was evaluated directly using a pull-down assay based on biotinylated cyclic-di-GMP and streptavidin-linked beads. IMPORTANCE The CckA, ChpT and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus. This work biochemically demonstrates that CckA, ChpT and CtrA also form a functional phosphorelay in this latter group, and that the direction of phosphotransfer is reversed by cyclic-di-GMP. It is important to improve the understanding of more representatives of this pathway to obtain a deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks.

scholarly journals Multistep Phosphorelay Proteins Transmit Oxidative Stress Signals to the Fission Yeast Stress-activated Protein Kinase

2000 ◽  
Vol 11 (4) ◽  
pp. 1169-1181 ◽  
Author(s):  
Aaron Ngocky Nguyen ◽  
Albert Lee ◽  
Warren Place ◽  
Kazuhiro Shiozaki
Keyword(s):  
Oxidative Stress ◽  
Protein Kinase ◽  
Response Regulator ◽  
Phosphorylation Site ◽  
Bzip Transcription Factor ◽  
Specific Gene ◽  
Putative Phosphorylation Site ◽  
Stress Signals ◽  
Cognate Response Regulator ◽  
Novel Protein

In response to oxidative stress, eukaryotic cells induce transcription of genes required for detoxification of oxidants. Here we present evidence that oxidative stress stimuli are transmitted by a multistep phosphorelay system to the Spc1/Sty1 stress-activated protein kinase in the fission yeastSchizosaccharomyces pombe. The fission yeastmpr1+gene encodes a novel protein with a histidine-containing phosphotransfer domain homologous to the budding yeast Ypd1. Spc1 activation upon oxidative stress is severely impaired in the Δmpr1 mutant as well as in thempr1HQ strain, in which the putative phosphorylation site Mpr1-His221 is substituted with glutamine. In response to oxidative stress, Mpr1 binds to the Mcs4 response regulator that functions upstream of the Spc1 cascade, suggesting that Mcs4 is a cognate response regulator for Mpr1. Unexpectedly, when exposed to hydrogen peroxide, Δmpr1 cells can induce the catalase gene ctt1+, one of the transcriptional targets of the Spc1 pathway, and survive oxidative stress in the absence of significant Spc1 activation. We have found that Pap1, a bZIP transcription factor homologous to human c-Jun, can mediate induction of ctt1+expression upon oxidative stress independently of the Spc1 stress-activated protein kinase. These studies show that oxidative stress stimuli are transmitted by multiple pathways to induce specific gene expression.

scholarly journals Characterization of novel mutations at the Schizosaccharomyces pombe cdc2 regulatory phosphorylation site, tyrosine 15.

1996 ◽  
Vol 7 (10) ◽  
pp. 1573-1586 ◽  
Author(s):  
K L Gould ◽  
A Feoktistova
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Phosphorylation Site ◽  
Loss Of Function ◽  
Contractile Ring ◽  
Mutant Proteins ◽  
Regulatory Phosphorylation ◽  
Binding Loop

The cdc2 protein kinase family is regulated negatively by phosphorylation in the glycine ATP-binding loop at a conserved tyrosine residue, Y15, alone or in combination with T14 phosphorylation. In Schizosaccharomyces pombe and other systems, substitution of these residues with structurally similar but nonphosphorylatable amino acids has generated proteins (Y15F or T14AY15F) that behave as constitutively tyrosine-dephosphorylated proteins or threonine and tyrosine-dephosphorylated proteins. Here we report the characteristics of three additional mutants at Y15--Y15E, Y15S, and Y15T--in S. pombe cdc2p. All three mutant proteins are active in in vitro kinase assays, but are unable to functionally complement cdc2 loss-of-function mutations in vivo. Additionally, all three mutants are dominant negatives. A more detailed analysis of the Y15T mutant indicates that it can initiate chromosome condensation and F-actin contractile ring formation, but is unable to drive the reorganization of microtubules into a mitotic spindle.

scholarly journals Regulation of p53 Function and Stability by Phosphorylation

1999 ◽  
Vol 19 (3) ◽  
pp. 1751-1758 ◽  
Author(s):  
Margaret Ashcroft ◽  
Michael H. G. Kubbutat ◽  
Karen H. Vousden
Keyword(s):  
Dna Damage ◽  
Protein Kinases ◽  
Phosphorylation Site ◽  
Tumor Suppressor Protein ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Mutant Proteins ◽  
Encoding Gene ◽  
P53 Tumor Suppressor Protein

ABSTRACT The p53 tumor suppressor protein can be phosphorylated at several sites within the N- and C-terminal domains, and several protein kinases have been shown to phosphorylate p53 in vitro. In this study, we examined the activity of p53 proteins with combined mutations at all of the reported N-terminal phosphorylation sites (p53N-term), all of the C-terminal phosphorylation sites (p53C-term), or all of the phosphorylation sites together (p53N/C-term). Each of these mutant proteins retained transcriptional transactivation functions, indicating that phosphorylation is not essential for this activity of p53, although a subtle contribution of the C-terminal phosphorylation sites to the activation of expression of the endogenous p21Waf1/Cip1-encoding gene was detected. Mutation of the phosphorylation sites to alanine did not affect the sensitivity of p53 to binding to or degradation by Mdm2, although alteration of residues 15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradation, consistent with recent reports that phosphorylation at these sites inhibits the p53-Mdm2 interaction. However, expression of the phosphorylation site mutant proteins in both wild-type p53-expressing and p53-null lines showed that all of the mutant proteins retained the ability to be stabilized following DNA damage. This indicates that phosphorylation is not essential for DNA damage-induced stabilization of p53, although phosphorylation could clearly contribute to p53 stabilization under some conditions.

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