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Multiple signals modulate the activity of the complex sensor kinase TodS.

Silva-Jiménez H, Ortega Á, García-Fontana C, Ramos JL, Krell T - Microb Biotechnol (2014)

Bottom Line: In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione.The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways.This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Protection, Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, C/ Prof. Albareda 1, Granada, 18008, Spain.

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Essential role of cysteines in the menadione-mediated regulation of TodS.A. Effect of alkylation on the kinase activity of TodS. TodS in the 2 mM DTT-containing analysis buffer was incubated with 1 mM NEM for 30 min prior to autophosphorylation assays in the presence of 0.1 mM toluene and different menadione concentrations.B. Autophosphorylation of TodS, TodSC110A and TodSC320A in the absence and presence of toluene. TodS (6.5 μM) were submitted to autophosphorylation assays in the absence and presence of 0.1 mM toluene.C. Effect of menadione on the autophosphorylation of TodS, TodSC110A and TodSC320A. Assays were carried out in 2 mM DTT- and 0.1 mM toluene-containing analysis buffer and different menadione concentrations.
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fig06: Essential role of cysteines in the menadione-mediated regulation of TodS.A. Effect of alkylation on the kinase activity of TodS. TodS in the 2 mM DTT-containing analysis buffer was incubated with 1 mM NEM for 30 min prior to autophosphorylation assays in the presence of 0.1 mM toluene and different menadione concentrations.B. Autophosphorylation of TodS, TodSC110A and TodSC320A in the absence and presence of toluene. TodS (6.5 μM) were submitted to autophosphorylation assays in the absence and presence of 0.1 mM toluene.C. Effect of menadione on the autophosphorylation of TodS, TodSC110A and TodSC320A. Assays were carried out in 2 mM DTT- and 0.1 mM toluene-containing analysis buffer and different menadione concentrations.

Mentions: Subsequent experiments were aimed at determining the role of cysteine residues in the menadione-mediated inhibition. TodS was treated with 1 mM N-ethylmaleimide (NEM), a reagent that modifies sulfhydryl groups with high specificity (Smyth et al., 1964; Paulech et al., 2013). Whereas NEM-untreated protein showed the expected menadione-dependent reduction in autophosphorylation (Fig. 6A), NEM-treated protein was inactive in the absence and presence of menadione, suggesting that cysteines play a central role in TodS activity.


Multiple signals modulate the activity of the complex sensor kinase TodS.

Silva-Jiménez H, Ortega Á, García-Fontana C, Ramos JL, Krell T - Microb Biotechnol (2014)

Essential role of cysteines in the menadione-mediated regulation of TodS.A. Effect of alkylation on the kinase activity of TodS. TodS in the 2 mM DTT-containing analysis buffer was incubated with 1 mM NEM for 30 min prior to autophosphorylation assays in the presence of 0.1 mM toluene and different menadione concentrations.B. Autophosphorylation of TodS, TodSC110A and TodSC320A in the absence and presence of toluene. TodS (6.5 μM) were submitted to autophosphorylation assays in the absence and presence of 0.1 mM toluene.C. Effect of menadione on the autophosphorylation of TodS, TodSC110A and TodSC320A. Assays were carried out in 2 mM DTT- and 0.1 mM toluene-containing analysis buffer and different menadione concentrations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321377&req=5

fig06: Essential role of cysteines in the menadione-mediated regulation of TodS.A. Effect of alkylation on the kinase activity of TodS. TodS in the 2 mM DTT-containing analysis buffer was incubated with 1 mM NEM for 30 min prior to autophosphorylation assays in the presence of 0.1 mM toluene and different menadione concentrations.B. Autophosphorylation of TodS, TodSC110A and TodSC320A in the absence and presence of toluene. TodS (6.5 μM) were submitted to autophosphorylation assays in the absence and presence of 0.1 mM toluene.C. Effect of menadione on the autophosphorylation of TodS, TodSC110A and TodSC320A. Assays were carried out in 2 mM DTT- and 0.1 mM toluene-containing analysis buffer and different menadione concentrations.
Mentions: Subsequent experiments were aimed at determining the role of cysteine residues in the menadione-mediated inhibition. TodS was treated with 1 mM N-ethylmaleimide (NEM), a reagent that modifies sulfhydryl groups with high specificity (Smyth et al., 1964; Paulech et al., 2013). Whereas NEM-untreated protein showed the expected menadione-dependent reduction in autophosphorylation (Fig. 6A), NEM-treated protein was inactive in the absence and presence of menadione, suggesting that cysteines play a central role in TodS activity.

Bottom Line: In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione.The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways.This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.

View Article: PubMed Central - PubMed

Affiliation: Department of Environmental Protection, Consejo Superior de Investigaciones Científicas, Estación Experimental del Zaidín, C/ Prof. Albareda 1, Granada, 18008, Spain.

Show MeSH
Related in: MedlinePlus