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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

DnaK Y601F exhibited impaired interaction with its co-chaperone proteins DnaJ and GrpE.(A) Sequence alignment of DnaKs from Bacillus subtilis, Lactobacillus casei, Thermoanaerobacter brockii, Clostridium difficile, Staphylococcus aureus, Streptococcus pneumonia, Escherichia coli, Desulfovibrio vulgaris, and Mycobacterium tuberculosis. Y601 in the B. subtilis DnaK is indicated by the arrow. (B) ATPase activity of DnaK WT and Y601F in three conditions: incubated alone, in the presence of DnaJ, and in the presence of both DnaJ and GrpE. The ATP hydrolysis activity of DnaJ and GrpE was subtracted from all relevant samples. The results are the mean values from five independent replicates, with error bars representing the standard deviation. ATPase activity is expressed as ATP hydrolysis rate/moles of the protein (μM min-1/nmol). (C) Interaction of DnaK WT/Y601F with DnaJ and GrpE detected by a pull-down assay. Experiment was performed with Strep-tagged DnaJ and GrpE, and 6xHis-tagged DnaK. DnaJ (lane 1) and GrpE (lane 5) were loaded directly on SDS-PAGE. DnaJ or GrpE incubated without DnaK (lanes 2 and 6), with DnaK WT (lanes 3 and 7), and with DnaK Y601F (lanes 4 and 8) were subjected to immunoprecipitation with an anti-6xHis antibody. The eluates from different samples were separated by SDS-PAGE, and detected by immunoblotting with anti-Strep tag and anti-His tag antibodies. The experiment was done in duplicates, and a representative image is shown.
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Figure 4: DnaK Y601F exhibited impaired interaction with its co-chaperone proteins DnaJ and GrpE.(A) Sequence alignment of DnaKs from Bacillus subtilis, Lactobacillus casei, Thermoanaerobacter brockii, Clostridium difficile, Staphylococcus aureus, Streptococcus pneumonia, Escherichia coli, Desulfovibrio vulgaris, and Mycobacterium tuberculosis. Y601 in the B. subtilis DnaK is indicated by the arrow. (B) ATPase activity of DnaK WT and Y601F in three conditions: incubated alone, in the presence of DnaJ, and in the presence of both DnaJ and GrpE. The ATP hydrolysis activity of DnaJ and GrpE was subtracted from all relevant samples. The results are the mean values from five independent replicates, with error bars representing the standard deviation. ATPase activity is expressed as ATP hydrolysis rate/moles of the protein (μM min-1/nmol). (C) Interaction of DnaK WT/Y601F with DnaJ and GrpE detected by a pull-down assay. Experiment was performed with Strep-tagged DnaJ and GrpE, and 6xHis-tagged DnaK. DnaJ (lane 1) and GrpE (lane 5) were loaded directly on SDS-PAGE. DnaJ or GrpE incubated without DnaK (lanes 2 and 6), with DnaK WT (lanes 3 and 7), and with DnaK Y601F (lanes 4 and 8) were subjected to immunoprecipitation with an anti-6xHis antibody. The eluates from different samples were separated by SDS-PAGE, and detected by immunoblotting with anti-Strep tag and anti-His tag antibodies. The experiment was done in duplicates, and a representative image is shown.

Mentions: The C-terminus of DnaK is known to be important for its interaction with its co-chaperone DnaJ (Gao et al., 2012). More specifically, the C-terminal helical subdomain downstream of the substrate-binding domain of DnaK is important for DnaJ binding, since the protein lacking this domain is unable to bind DnaJ. Recently, a conserved motif DXXXEEV was identified in the extreme C-terminal tail of bacterial DnaKs. In E. coli, this motif was shown to be crucial for in vivo cell survival upon heat shock and in vitro chaperone activity of DnaK. Moreover, the DXXXEEV motif was predicted to be a potential protein binding region (Smock et al., 2011). Interestingly, Y601 in B. subtilis DnaK is situated within this motif (Figure 4A). We examined 934 available sequences of bacterial DnaKs, and found Y601 to be conserved in 20.88% of them, and in 25.35% of DnaKs from Firmicutes. The most common residue at this position is phenylalanine, which is a non-phosphorylatable version of tyrosine. Phenylalanine is found at the position equivalent to B. subtilis Y601 in 50.43% of all bacterial DnaKs, and 66.55% of DnaKs from Firmicutes. Reversible phosphorylation of tyrosine Y601 adds a negatively charged residue in this DXXXEEV motif (DAEYEEV in B. subtilis), and could thus be expected to have an impact on DnaK function. One of the known consequences of DnaK interaction with DnaJ is the enhancement of the ATP hydrolysis catalyzed by DnaK (Laufen et al., 1999). Therefore we asked whether DnaJ would be capable of enhancing the ATPase activity of DnaK Y601F. As shown in the Figure 4B, DnaK Y601F exhibited a slightly impaired ATPase activity compared to the WT. More interestingly, neither DnaJ, or DnaJ and GrpE together, provided any detectable enhancement of the ATPase activity of DnaK Y601F. Since DnaJ and GrpE stimulate DnaK ATPase activity via a direct protein–protein interaction, DnaK Y601F should be expected to have an impaired interaction with its co-chaperones. To test the interaction of DnaK with DnaJ and GrpE we used a pull-down assay (Figure 4C). Binary interactions of DnaK with DnaJ and GrpE were readily detectable. Mutated protein DnaK Y601F had a clearly diminished capacity to interact with both co-chaperones, indicating that residue tyrosine 601 indeed contributes to the interactions. Thus, we concluded that tyrosine 601 of DnaK is important to its interaction with co-chaperones DnaJ and GrpE, and consequently to activation of its ATPase function.


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 exhibited impaired interaction with its co-chaperone proteins DnaJ and GrpE.(A) Sequence alignment of DnaKs from Bacillus subtilis, Lactobacillus casei, Thermoanaerobacter brockii, Clostridium difficile, Staphylococcus aureus, Streptococcus pneumonia, Escherichia coli, Desulfovibrio vulgaris, and Mycobacterium tuberculosis. Y601 in the B. subtilis DnaK is indicated by the arrow. (B) ATPase activity of DnaK WT and Y601F in three conditions: incubated alone, in the presence of DnaJ, and in the presence of both DnaJ and GrpE. The ATP hydrolysis activity of DnaJ and GrpE was subtracted from all relevant samples. The results are the mean values from five independent replicates, with error bars representing the standard deviation. ATPase activity is expressed as ATP hydrolysis rate/moles of the protein (μM min-1/nmol). (C) Interaction of DnaK WT/Y601F with DnaJ and GrpE detected by a pull-down assay. Experiment was performed with Strep-tagged DnaJ and GrpE, and 6xHis-tagged DnaK. DnaJ (lane 1) and GrpE (lane 5) were loaded directly on SDS-PAGE. DnaJ or GrpE incubated without DnaK (lanes 2 and 6), with DnaK WT (lanes 3 and 7), and with DnaK Y601F (lanes 4 and 8) were subjected to immunoprecipitation with an anti-6xHis antibody. The eluates from different samples were separated by SDS-PAGE, and detected by immunoblotting with anti-Strep tag and anti-His tag antibodies. The experiment was done in duplicates, and a representative image is shown.
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Figure 4: DnaK Y601F exhibited impaired interaction with its co-chaperone proteins DnaJ and GrpE.(A) Sequence alignment of DnaKs from Bacillus subtilis, Lactobacillus casei, Thermoanaerobacter brockii, Clostridium difficile, Staphylococcus aureus, Streptococcus pneumonia, Escherichia coli, Desulfovibrio vulgaris, and Mycobacterium tuberculosis. Y601 in the B. subtilis DnaK is indicated by the arrow. (B) ATPase activity of DnaK WT and Y601F in three conditions: incubated alone, in the presence of DnaJ, and in the presence of both DnaJ and GrpE. The ATP hydrolysis activity of DnaJ and GrpE was subtracted from all relevant samples. The results are the mean values from five independent replicates, with error bars representing the standard deviation. ATPase activity is expressed as ATP hydrolysis rate/moles of the protein (μM min-1/nmol). (C) Interaction of DnaK WT/Y601F with DnaJ and GrpE detected by a pull-down assay. Experiment was performed with Strep-tagged DnaJ and GrpE, and 6xHis-tagged DnaK. DnaJ (lane 1) and GrpE (lane 5) were loaded directly on SDS-PAGE. DnaJ or GrpE incubated without DnaK (lanes 2 and 6), with DnaK WT (lanes 3 and 7), and with DnaK Y601F (lanes 4 and 8) were subjected to immunoprecipitation with an anti-6xHis antibody. The eluates from different samples were separated by SDS-PAGE, and detected by immunoblotting with anti-Strep tag and anti-His tag antibodies. The experiment was done in duplicates, and a representative image is shown.
Mentions: The C-terminus of DnaK is known to be important for its interaction with its co-chaperone DnaJ (Gao et al., 2012). More specifically, the C-terminal helical subdomain downstream of the substrate-binding domain of DnaK is important for DnaJ binding, since the protein lacking this domain is unable to bind DnaJ. Recently, a conserved motif DXXXEEV was identified in the extreme C-terminal tail of bacterial DnaKs. In E. coli, this motif was shown to be crucial for in vivo cell survival upon heat shock and in vitro chaperone activity of DnaK. Moreover, the DXXXEEV motif was predicted to be a potential protein binding region (Smock et al., 2011). Interestingly, Y601 in B. subtilis DnaK is situated within this motif (Figure 4A). We examined 934 available sequences of bacterial DnaKs, and found Y601 to be conserved in 20.88% of them, and in 25.35% of DnaKs from Firmicutes. The most common residue at this position is phenylalanine, which is a non-phosphorylatable version of tyrosine. Phenylalanine is found at the position equivalent to B. subtilis Y601 in 50.43% of all bacterial DnaKs, and 66.55% of DnaKs from Firmicutes. Reversible phosphorylation of tyrosine Y601 adds a negatively charged residue in this DXXXEEV motif (DAEYEEV in B. subtilis), and could thus be expected to have an impact on DnaK function. One of the known consequences of DnaK interaction with DnaJ is the enhancement of the ATP hydrolysis catalyzed by DnaK (Laufen et al., 1999). Therefore we asked whether DnaJ would be capable of enhancing the ATPase activity of DnaK Y601F. As shown in the Figure 4B, DnaK Y601F exhibited a slightly impaired ATPase activity compared to the WT. More interestingly, neither DnaJ, or DnaJ and GrpE together, provided any detectable enhancement of the ATPase activity of DnaK Y601F. Since DnaJ and GrpE stimulate DnaK ATPase activity via a direct protein–protein interaction, DnaK Y601F should be expected to have an impaired interaction with its co-chaperones. To test the interaction of DnaK with DnaJ and GrpE we used a pull-down assay (Figure 4C). Binary interactions of DnaK with DnaJ and GrpE were readily detectable. Mutated protein DnaK Y601F had a clearly diminished capacity to interact with both co-chaperones, indicating that residue tyrosine 601 indeed contributes to the interactions. Thus, we concluded that tyrosine 601 of DnaK is important to its interaction with co-chaperones DnaJ and GrpE, and consequently to activation of its ATPase function.

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