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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

Stable Isotope Labeling by Amino acids in Cell culture (SILAC) screen: DnaK phosphorylation at Y601 depends on PtkA and PtpZ.(A) Scatter plots showing ΔptkA/WT and ΔptpZ/WT SILAC ratios, respectively. Log2 ratios of the phosphorylation sites are normalized to the corresponding protein and plotted against peptide intensities in the log10 scale. Significantly changing (p = 0.05) SILAC ratios marked in red present potential substrates of PtkA (M/L Ratios) and PtpZ (H/L Ratios), respectively. The spot corresponding to DnaK Y601 is marked with a red circle. (B) Spectrum of the DnaK peptide containing phosphorylation at Y601. The SILAC triplet of the DnaK peptide obtained during the survey scan is shown above. The MS/MS spectrum of the DnaK peptide depicting the phosphorylation event on Y601 is shown below.
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Figure 1: Stable Isotope Labeling by Amino acids in Cell culture (SILAC) screen: DnaK phosphorylation at Y601 depends on PtkA and PtpZ.(A) Scatter plots showing ΔptkA/WT and ΔptpZ/WT SILAC ratios, respectively. Log2 ratios of the phosphorylation sites are normalized to the corresponding protein and plotted against peptide intensities in the log10 scale. Significantly changing (p = 0.05) SILAC ratios marked in red present potential substrates of PtkA (M/L Ratios) and PtpZ (H/L Ratios), respectively. The spot corresponding to DnaK Y601 is marked with a red circle. (B) Spectrum of the DnaK peptide containing phosphorylation at Y601. The SILAC triplet of the DnaK peptide obtained during the survey scan is shown above. The MS/MS spectrum of the DnaK peptide depicting the phosphorylation event on Y601 is shown below.

Mentions: The global phosphoproteome analysis of knockout strains, followed by normalization against the proteome, led to the detection of six differentially down-regulated phosphorylation events in the ΔptkA strain and four differentially up-regulated events in the ΔptpZ strain (Figure 1A). Three phosphotyrosine sites of PtkA- Y225, Y227, and Y228, were detected as down-regulated in the ΔptkA strain. They are known PtkA autophosphorylation sites (Mijakovic et al., 2003). Although these three sites were absent in the ΔptkA strain, this outcome is a result of an artifact of normalization with the WT control strain. Y225, Y227, and Y228 of PtkA are also known to be dephosphorylated by PtpZ (Mijakovic et al., 2003), and they were duly detected as up-regulated in the ΔptpZ, thus acting as a positive control. Phosphorylation of another B. subtilis BY-kinase, PtkB (YveL, EpsB), at the residue Y227, was down-regulated in the ΔptkA strain. PtkB plays a role in production of exopolysaccharides (Elsholz et al., 2014) and biofilm development (Gerwig et al., 2014). It is known that some level of interplay between PtkA and PtkB exists, since the two kinases seem to be able to swap their respective transmembrane activators, TkmA and TkmB (Shi et al., 2014c). This result suggests that in addition, PtkA may be able to phosphorylate PtkB, or otherwise indirectly contribute to its autophosphorylation at the residue Y227. Interestingly, phosphorylation of PtkB was not up-regulated in the ΔptpZ strain, suggesting that this phosphatase is not responsible for its dephosphorylation. Among other PtkA- and PtpZ-dependent phosphoproteins were SunI and DnaK. Phosphorylation of both SunI and DnaK was down-regulated in ΔptkA and up-regulated in ΔptpZ. SunI, a bacteriocin producer immunity protein that plays an important role in conferring immunity to the bacterium against sublancin, was phosphorylated at Y57. DnaK, a class-I heat-shock protein that acts as a molecular chaperone, was detected to be phosphorylated at Y601. It is important to remark that in this study we have detected 47 tyrosine-phosphorylation sites (Table 1), only eight of which have been detected previously. The result suggests that tyrosine phosphorylation is involved in multiple cellular functions: the development processes such as sporulation (CotH and SdpB) and biofilm formation (PtkB), competence (ComFB and ComGB), biosynthesis (AcoB, CatE, Fbp, MgsA, ThiC, ThrC, and Ugd), cellular regulation (RsbV, PtkA, YbfP, and SasA) and other physiological processes (DnaK, PbpD, RplA, YqiB, YkoV, YocA, SunI, and YxiD). Ugd, which had previously been found to be phosphorylated at Y81 by PtkA (Petranovic et al., 2009), was now found to be phosphorylated also at Y100.


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)

Stable Isotope Labeling by Amino acids in Cell culture (SILAC) screen: DnaK phosphorylation at Y601 depends on PtkA and PtpZ.(A) Scatter plots showing ΔptkA/WT and ΔptpZ/WT SILAC ratios, respectively. Log2 ratios of the phosphorylation sites are normalized to the corresponding protein and plotted against peptide intensities in the log10 scale. Significantly changing (p = 0.05) SILAC ratios marked in red present potential substrates of PtkA (M/L Ratios) and PtpZ (H/L Ratios), respectively. The spot corresponding to DnaK Y601 is marked with a red circle. (B) Spectrum of the DnaK peptide containing phosphorylation at Y601. The SILAC triplet of the DnaK peptide obtained during the survey scan is shown above. The MS/MS spectrum of the DnaK peptide depicting the phosphorylation event on Y601 is shown below.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Stable Isotope Labeling by Amino acids in Cell culture (SILAC) screen: DnaK phosphorylation at Y601 depends on PtkA and PtpZ.(A) Scatter plots showing ΔptkA/WT and ΔptpZ/WT SILAC ratios, respectively. Log2 ratios of the phosphorylation sites are normalized to the corresponding protein and plotted against peptide intensities in the log10 scale. Significantly changing (p = 0.05) SILAC ratios marked in red present potential substrates of PtkA (M/L Ratios) and PtpZ (H/L Ratios), respectively. The spot corresponding to DnaK Y601 is marked with a red circle. (B) Spectrum of the DnaK peptide containing phosphorylation at Y601. The SILAC triplet of the DnaK peptide obtained during the survey scan is shown above. The MS/MS spectrum of the DnaK peptide depicting the phosphorylation event on Y601 is shown below.
Mentions: The global phosphoproteome analysis of knockout strains, followed by normalization against the proteome, led to the detection of six differentially down-regulated phosphorylation events in the ΔptkA strain and four differentially up-regulated events in the ΔptpZ strain (Figure 1A). Three phosphotyrosine sites of PtkA- Y225, Y227, and Y228, were detected as down-regulated in the ΔptkA strain. They are known PtkA autophosphorylation sites (Mijakovic et al., 2003). Although these three sites were absent in the ΔptkA strain, this outcome is a result of an artifact of normalization with the WT control strain. Y225, Y227, and Y228 of PtkA are also known to be dephosphorylated by PtpZ (Mijakovic et al., 2003), and they were duly detected as up-regulated in the ΔptpZ, thus acting as a positive control. Phosphorylation of another B. subtilis BY-kinase, PtkB (YveL, EpsB), at the residue Y227, was down-regulated in the ΔptkA strain. PtkB plays a role in production of exopolysaccharides (Elsholz et al., 2014) and biofilm development (Gerwig et al., 2014). It is known that some level of interplay between PtkA and PtkB exists, since the two kinases seem to be able to swap their respective transmembrane activators, TkmA and TkmB (Shi et al., 2014c). This result suggests that in addition, PtkA may be able to phosphorylate PtkB, or otherwise indirectly contribute to its autophosphorylation at the residue Y227. Interestingly, phosphorylation of PtkB was not up-regulated in the ΔptpZ strain, suggesting that this phosphatase is not responsible for its dephosphorylation. Among other PtkA- and PtpZ-dependent phosphoproteins were SunI and DnaK. Phosphorylation of both SunI and DnaK was down-regulated in ΔptkA and up-regulated in ΔptpZ. SunI, a bacteriocin producer immunity protein that plays an important role in conferring immunity to the bacterium against sublancin, was phosphorylated at Y57. DnaK, a class-I heat-shock protein that acts as a molecular chaperone, was detected to be phosphorylated at Y601. It is important to remark that in this study we have detected 47 tyrosine-phosphorylation sites (Table 1), only eight of which have been detected previously. The result suggests that tyrosine phosphorylation is involved in multiple cellular functions: the development processes such as sporulation (CotH and SdpB) and biofilm formation (PtkB), competence (ComFB and ComGB), biosynthesis (AcoB, CatE, Fbp, MgsA, ThiC, ThrC, and Ugd), cellular regulation (RsbV, PtkA, YbfP, and SasA) and other physiological processes (DnaK, PbpD, RplA, YqiB, YkoV, YocA, SunI, and YxiD). Ugd, which had previously been found to be phosphorylated at Y81 by PtkA (Petranovic et al., 2009), was now found to be phosphorylated also at Y100.

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