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Tyrosine 601 of Bacillus subtilis DnaK Undergoes Phosphorylation and Is Crucial for Chaperone Activity and Heat Shock Survival.

Shi L, Ravikumar V, Derouiche A, Macek B, Mijakovic I - Front Microbiol (2016)

Bottom Line: The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the WT dnaK allele expressed ectopically.We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively.Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis.

View Article: PubMed Central - PubMed

Affiliation: Division of Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology Gothenburg, Sweden.

ABSTRACT
In order to screen for cellular substrates of the Bacillus subtilis BY-kinase PtkA, and its cognate phosphotyrosine-protein phosphatase PtpZ, we performed a triple Stable Isotope Labeling by Amino acids in Cell culture-based quantitative phosphoproteome analysis. Detected tyrosine phosphorylation sites for which the phosphorylation level decreased in the ΔptkA strain and increased in the ΔptpZ strain, compared to the wild type (WT), were considered as potential substrates of PtkA/PtpZ. One of those sites was the residue tyrosine 601 of the molecular chaperone DnaK. We confirmed that DnaK is a substrate of PtkA and PtpZ by in vitro phosphorylation and dephosphorylation assays. In vitro, DnaK Y601F mutant exhibited impaired interaction with its co-chaperones DnaJ and GrpE, along with diminished capacity to hydrolyze ATP and assist the re-folding of denatured proteins. In vivo, loss of DnaK phosphorylation in the mutant strain dnaK Y601F, or in the strain overexpressing the phosphatase PtpZ, led to diminished survival upon heat shock, consistent with the in vitro results. The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the WT dnaK allele expressed ectopically. We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively. Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis.

No MeSH data available.


Related in: MedlinePlus

Non-phosphorylated state of DnaK results in impaired heat resistance in vivo.(A) Survival of the heat shock treatment (55°C for 5 min) of B. subtilis strains ΔptkAΔlysA and dnaK Y601F compared to their respective WT. Heat resistance is expressed as the number of CFUs after treatment/number of CFUs without treatment. Each strain was normalized with respect to the WT. The results are the mean values from three independent replicates, with error bars representing the standard deviation. (B) Growth tests for B. subtilis strain: WT, ptpZ overexpression, Y601F and Y601F carrying WT copy of dnaK. Serial dilutions of each culture were spotted onto plates and incubated at 58°C for 10 h and additional 4 h at 37°C. The result is representative of three independent replicates.
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Figure 6: Non-phosphorylated state of DnaK results in impaired heat resistance in vivo.(A) Survival of the heat shock treatment (55°C for 5 min) of B. subtilis strains ΔptkAΔlysA and dnaK Y601F compared to their respective WT. Heat resistance is expressed as the number of CFUs after treatment/number of CFUs without treatment. Each strain was normalized with respect to the WT. The results are the mean values from three independent replicates, with error bars representing the standard deviation. (B) Growth tests for B. subtilis strain: WT, ptpZ overexpression, Y601F and Y601F carrying WT copy of dnaK. Serial dilutions of each culture were spotted onto plates and incubated at 58°C for 10 h and additional 4 h at 37°C. The result is representative of three independent replicates.

Mentions: In order to investigate the effect of DnaK tyrosine 601 in vivo, we subjected dnaK Y601F (point mutation at the locus) to a 5 min heat shock at 55°C, and measured the fraction of surviving cells. Compared to the WT strain, dnaK Y601F provoked a significant drop in survival (Figure 6A). The result indicated the function importance of DnaK tyrosine 601 in vivo. The involvement of the tyrosine 601 in DnaK function could be trigger by the hydroxyl group itself, or alternatively trough phosphorylation. We subjected the strains ΔptkAΔlysA (used for the triple SILAC experiment) to the same heat resistance assay. The inactivation of PtkA led to a reduced survival upon heat shock in vivo (Figure 6A), which suggests the regulatory potential of this phosphorylation event. The phosphoproteome and in vitro dephosphorylation data suggested that PtpZ is the phosphatase responsible for DnaK dephosphorylation (Figures 1A and 2A). To test this assumption in vivo, we overproduced PtpZ. The strain overproducing PtpZ showed decreased ability to grow at elevated temperature (Figure 6B), consistent with it having less phosphorylated DnaK. In the same assay, the strain with the dnaK Y601F mutation had a more severe phenotype, which suggests that overexpressed PtpZ could not entirely dephosphorylate the tyrosine 601 in vivo. The severe phenotype of the dnaK Y601F mutation could be complemented by two orders of magnitude (in terms of survival/dilution) by expressing the WT dnaK allele ectopically (Figure 6B). These results suggested that the tyrosine 601 of DnaK is crucial for the cellular survival upon heat shock, and the most likely mechanism of its contribution to DnaK chaperone function is through PtkA-dependent phosphorylation and PtpZ-dependent dephosphorylation.


Tyrosine 601 of Bacillus subtilis DnaK Undergoes Phosphorylation and Is Crucial for Chaperone Activity and Heat Shock Survival.

Shi L, Ravikumar V, Derouiche A, Macek B, Mijakovic I - Front Microbiol (2016)

Non-phosphorylated state of DnaK results in impaired heat resistance in vivo.(A) Survival of the heat shock treatment (55°C for 5 min) of B. subtilis strains ΔptkAΔlysA and dnaK Y601F compared to their respective WT. Heat resistance is expressed as the number of CFUs after treatment/number of CFUs without treatment. Each strain was normalized with respect to the WT. The results are the mean values from three independent replicates, with error bars representing the standard deviation. (B) Growth tests for B. subtilis strain: WT, ptpZ overexpression, Y601F and Y601F carrying WT copy of dnaK. Serial dilutions of each culture were spotted onto plates and incubated at 58°C for 10 h and additional 4 h at 37°C. The result is representative of three independent replicates.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835898&req=5

Figure 6: Non-phosphorylated state of DnaK results in impaired heat resistance in vivo.(A) Survival of the heat shock treatment (55°C for 5 min) of B. subtilis strains ΔptkAΔlysA and dnaK Y601F compared to their respective WT. Heat resistance is expressed as the number of CFUs after treatment/number of CFUs without treatment. Each strain was normalized with respect to the WT. The results are the mean values from three independent replicates, with error bars representing the standard deviation. (B) Growth tests for B. subtilis strain: WT, ptpZ overexpression, Y601F and Y601F carrying WT copy of dnaK. Serial dilutions of each culture were spotted onto plates and incubated at 58°C for 10 h and additional 4 h at 37°C. The result is representative of three independent replicates.
Mentions: In order to investigate the effect of DnaK tyrosine 601 in vivo, we subjected dnaK Y601F (point mutation at the locus) to a 5 min heat shock at 55°C, and measured the fraction of surviving cells. Compared to the WT strain, dnaK Y601F provoked a significant drop in survival (Figure 6A). The result indicated the function importance of DnaK tyrosine 601 in vivo. The involvement of the tyrosine 601 in DnaK function could be trigger by the hydroxyl group itself, or alternatively trough phosphorylation. We subjected the strains ΔptkAΔlysA (used for the triple SILAC experiment) to the same heat resistance assay. The inactivation of PtkA led to a reduced survival upon heat shock in vivo (Figure 6A), which suggests the regulatory potential of this phosphorylation event. The phosphoproteome and in vitro dephosphorylation data suggested that PtpZ is the phosphatase responsible for DnaK dephosphorylation (Figures 1A and 2A). To test this assumption in vivo, we overproduced PtpZ. The strain overproducing PtpZ showed decreased ability to grow at elevated temperature (Figure 6B), consistent with it having less phosphorylated DnaK. In the same assay, the strain with the dnaK Y601F mutation had a more severe phenotype, which suggests that overexpressed PtpZ could not entirely dephosphorylate the tyrosine 601 in vivo. The severe phenotype of the dnaK Y601F mutation could be complemented by two orders of magnitude (in terms of survival/dilution) by expressing the WT dnaK allele ectopically (Figure 6B). These results suggested that the tyrosine 601 of DnaK is crucial for the cellular survival upon heat shock, and the most likely mechanism of its contribution to DnaK chaperone function is through PtkA-dependent phosphorylation and PtpZ-dependent dephosphorylation.

Bottom Line: The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the WT dnaK allele expressed ectopically.We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively.Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis.

View Article: PubMed Central - PubMed

Affiliation: Division of Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of Technology Gothenburg, Sweden.

ABSTRACT
In order to screen for cellular substrates of the Bacillus subtilis BY-kinase PtkA, and its cognate phosphotyrosine-protein phosphatase PtpZ, we performed a triple Stable Isotope Labeling by Amino acids in Cell culture-based quantitative phosphoproteome analysis. Detected tyrosine phosphorylation sites for which the phosphorylation level decreased in the ΔptkA strain and increased in the ΔptpZ strain, compared to the wild type (WT), were considered as potential substrates of PtkA/PtpZ. One of those sites was the residue tyrosine 601 of the molecular chaperone DnaK. We confirmed that DnaK is a substrate of PtkA and PtpZ by in vitro phosphorylation and dephosphorylation assays. In vitro, DnaK Y601F mutant exhibited impaired interaction with its co-chaperones DnaJ and GrpE, along with diminished capacity to hydrolyze ATP and assist the re-folding of denatured proteins. In vivo, loss of DnaK phosphorylation in the mutant strain dnaK Y601F, or in the strain overexpressing the phosphatase PtpZ, led to diminished survival upon heat shock, consistent with the in vitro results. The decreased survival of the mutant dnaK Y601F at an elevated temperature could be rescued by complementing with the WT dnaK allele expressed ectopically. We concluded that the residue tyrosine 601 of DnaK can be phosphorylated and dephosphorylated by PtkA and PtpZ, respectively. Furthermore, Y601 is important for DnaK chaperone activity and heat shock survival of B. subtilis.

No MeSH data available.


Related in: MedlinePlus