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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.

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DnaK Y601F is less efficient in chaperone activity.(A) Refolding of GuHCl-denatured LDH by DnaK WT and Y601F. LDH: activity of LDH without GuHCl treatment; WT: activity of denatured LDH in the presence of DnaK WT; Y601F: activity of denatured LDH in the presence of DnaK Y601F; deLDH: activity of denatured LDH in the absence of DnaK. (B) Heat resistance of DnaK WT and Y601F to a heat treatment at 65°C for 5 min. DnaK ATP hydrolysis activity is expressed as a ratio of activity after and before the heat treatment. DnaK Y601F was normalized with respect to the WT. The results are the mean values from five independent replicates, with error bars representing the standard deviation.
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Figure 5: DnaK Y601F is less efficient in chaperone activity.(A) Refolding of GuHCl-denatured LDH by DnaK WT and Y601F. LDH: activity of LDH without GuHCl treatment; WT: activity of denatured LDH in the presence of DnaK WT; Y601F: activity of denatured LDH in the presence of DnaK Y601F; deLDH: activity of denatured LDH in the absence of DnaK. (B) Heat resistance of DnaK WT and Y601F to a heat treatment at 65°C for 5 min. DnaK ATP hydrolysis activity is expressed as a ratio of activity after and before the heat treatment. DnaK Y601F was normalized with respect to the WT. The results are the mean values from five independent replicates, with error bars representing the standard deviation.

Mentions: One of the major roles of molecular chaperones is to facilitate refolding of denatured proteins at high temperatures. Based on the in vitro data presented in the previous section, we concluded that DnaK tyrosine 601 is crucial for its interaction with co-chaperones and its ATPase function, both of which contribute to its activity as a molecular chaperone. Therefore, we asked whether the mutation on tyrosine 601 would have a measurable impact on its ability to re-fold denatured proteins. To test this, we established an assay with LDH denatured with a chaotropic agent. The activity of DnaK was measured as its capacity to restore the enzyme activity of denatured LDH (Figure 5A). WT DnaK, was able to restore 75% of the original activity of the LDH in 50 min. The Y601F mutant of DnaK was less efficient, and restored only about 50% of the original LDH activity in the same period. Thus, we concluded that the tyrosine 601 is important for the maintenance of the DnaK chaperone activity. In order to accomplish their role, molecular chaperones must themselves remain active under denaturing conditions. Therefore, we asked whether the DnaK Y601F remains stable under heat shock. To answer this, we measured the conservation of the ATPase activity of DnaK at high temperature in vitro (Figure 5B). The results are expressed as the ratio of ATPase activity of the heat-treated protein to the non-treated protein. DnaK Y601F exhibited a more severe loss of activity than the WT, indicating an impaired capacity to tolerate high temperature. We hypothesize that the Y601 could be involved in the regulation of DnaK chaperone activity through its phosphorylation. Previous findings in E. coli suggested chaperones became phosphorylated during heat shock (Sherman and Goldberg, 1992, 1993). The phosphorylated fraction of E. coli chaperones exhibited an increased capacity to interact with and re-fold denatured proteins. This is in agreement with the fact that DnaK WT, partially phosphorylated during production in E. coli, shows higher chaperone activity. However, we could not exclude the possibility that the impaired chaperone function of DnaK Y601F was caused by the lack of the free hydroxyl group, rather than by phosphorylation itself. An example of such a scenario is phosphorylation of the B. subtilis FatR, a transcriptional regulator involved in regulation of polyunsaturated fatty acids metabolism. FatR forms dimers, which bind the target DNA. The hydroxyl group of its tyrosine 45 is essential for the protein–DNA interaction, so phosphorylation basically had the same effect as removing the hydroxyl by replacing tyrosine 45t with phenylalanine, namely the loss of interaction with DNA (Derouiche et al., 2013).


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)

DnaK Y601F is less efficient in chaperone activity.(A) Refolding of GuHCl-denatured LDH by DnaK WT and Y601F. LDH: activity of LDH without GuHCl treatment; WT: activity of denatured LDH in the presence of DnaK WT; Y601F: activity of denatured LDH in the presence of DnaK Y601F; deLDH: activity of denatured LDH in the absence of DnaK. (B) Heat resistance of DnaK WT and Y601F to a heat treatment at 65°C for 5 min. DnaK ATP hydrolysis activity is expressed as a ratio of activity after and before the heat treatment. DnaK Y601F was normalized with respect to the WT. The results are the mean values from five independent replicates, with error bars representing the standard deviation.
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Related In: Results  -  Collection

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Figure 5: DnaK Y601F is less efficient in chaperone activity.(A) Refolding of GuHCl-denatured LDH by DnaK WT and Y601F. LDH: activity of LDH without GuHCl treatment; WT: activity of denatured LDH in the presence of DnaK WT; Y601F: activity of denatured LDH in the presence of DnaK Y601F; deLDH: activity of denatured LDH in the absence of DnaK. (B) Heat resistance of DnaK WT and Y601F to a heat treatment at 65°C for 5 min. DnaK ATP hydrolysis activity is expressed as a ratio of activity after and before the heat treatment. DnaK Y601F was normalized with respect to the WT. The results are the mean values from five independent replicates, with error bars representing the standard deviation.
Mentions: One of the major roles of molecular chaperones is to facilitate refolding of denatured proteins at high temperatures. Based on the in vitro data presented in the previous section, we concluded that DnaK tyrosine 601 is crucial for its interaction with co-chaperones and its ATPase function, both of which contribute to its activity as a molecular chaperone. Therefore, we asked whether the mutation on tyrosine 601 would have a measurable impact on its ability to re-fold denatured proteins. To test this, we established an assay with LDH denatured with a chaotropic agent. The activity of DnaK was measured as its capacity to restore the enzyme activity of denatured LDH (Figure 5A). WT DnaK, was able to restore 75% of the original activity of the LDH in 50 min. The Y601F mutant of DnaK was less efficient, and restored only about 50% of the original LDH activity in the same period. Thus, we concluded that the tyrosine 601 is important for the maintenance of the DnaK chaperone activity. In order to accomplish their role, molecular chaperones must themselves remain active under denaturing conditions. Therefore, we asked whether the DnaK Y601F remains stable under heat shock. To answer this, we measured the conservation of the ATPase activity of DnaK at high temperature in vitro (Figure 5B). The results are expressed as the ratio of ATPase activity of the heat-treated protein to the non-treated protein. DnaK Y601F exhibited a more severe loss of activity than the WT, indicating an impaired capacity to tolerate high temperature. We hypothesize that the Y601 could be involved in the regulation of DnaK chaperone activity through its phosphorylation. Previous findings in E. coli suggested chaperones became phosphorylated during heat shock (Sherman and Goldberg, 1992, 1993). The phosphorylated fraction of E. coli chaperones exhibited an increased capacity to interact with and re-fold denatured proteins. This is in agreement with the fact that DnaK WT, partially phosphorylated during production in E. coli, shows higher chaperone activity. However, we could not exclude the possibility that the impaired chaperone function of DnaK Y601F was caused by the lack of the free hydroxyl group, rather than by phosphorylation itself. An example of such a scenario is phosphorylation of the B. subtilis FatR, a transcriptional regulator involved in regulation of polyunsaturated fatty acids metabolism. FatR forms dimers, which bind the target DNA. The hydroxyl group of its tyrosine 45 is essential for the protein–DNA interaction, so phosphorylation basically had the same effect as removing the hydroxyl by replacing tyrosine 45t with phenylalanine, namely the loss of interaction with DNA (Derouiche et al., 2013).

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