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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

Y601 of DnaK is phosphorylated by PtkA and dephosphorylated by PtpZ in vitro. Bands corresponding to phosphorylated DnaK and PtkA are indicated by arrows. (A) Phosphorylation and dephosphorylation assays by Pro-Q Diamond Phosphoprotein Gel Staining. In lane 1–5, reactions were performed with 1.2 μM PtkA, 1.2 μM TkmA, 3.6 μM DnaK WT, or Y601F, and incubated for 30 min. Presence of key proteins in the assays is indicated with ± above each lane. In lanes 6–8, 5 μM PtpZ was added into pre-incubated reaction containing 0.6 μM PtkA, 0.6 μM TkmA, and 3.6 μM DnaK WT. The incubation times are given above each lane. (B) Autoradiography images of the phosphorylation of DnaK WT (lanes 2–4) and Y601F (lanes 5–7) in the presence of PtkA, and dephosphorylating of DnaK WT (lanes 8–11) in the presence of PtpZ. Phosphorylation assay was performed with 1 μM PtkA, 1 μM TkmA, and 3 μM DnaK. PtkA was incubated alone in the lane 1. In dephosphorylation assay, 5 μM PtpZ was added into pre-incubated reaction containing 1 μM PtkA, 1 μM TkmA, and 3 μM DnaK WT. The incubation times are given above each lane.
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Figure 3: Y601 of DnaK is phosphorylated by PtkA and dephosphorylated by PtpZ in vitro. Bands corresponding to phosphorylated DnaK and PtkA are indicated by arrows. (A) Phosphorylation and dephosphorylation assays by Pro-Q Diamond Phosphoprotein Gel Staining. In lane 1–5, reactions were performed with 1.2 μM PtkA, 1.2 μM TkmA, 3.6 μM DnaK WT, or Y601F, and incubated for 30 min. Presence of key proteins in the assays is indicated with ± above each lane. In lanes 6–8, 5 μM PtpZ was added into pre-incubated reaction containing 0.6 μM PtkA, 0.6 μM TkmA, and 3.6 μM DnaK WT. The incubation times are given above each lane. (B) Autoradiography images of the phosphorylation of DnaK WT (lanes 2–4) and Y601F (lanes 5–7) in the presence of PtkA, and dephosphorylating of DnaK WT (lanes 8–11) in the presence of PtpZ. Phosphorylation assay was performed with 1 μM PtkA, 1 μM TkmA, and 3 μM DnaK. PtkA was incubated alone in the lane 1. In dephosphorylation assay, 5 μM PtpZ was added into pre-incubated reaction containing 1 μM PtkA, 1 μM TkmA, and 3 μM DnaK WT. The incubation times are given above each lane.

Mentions: Following the triple SILAC screening, we set out to explore the functional relevance of one differentially phosphorylated phosphotyrosine-site, Y601 of DnaK. Manual validation of the MS/MS spectrum of the phosphorylated DnaK peptide showed good coverage and annotation of fragment ions (Figure 1B). In order to assess whether DnaK can be phosphorylated by PtkA and dephosphorylated by PtpZ, we proceeded with an in vitro phosphorylation/dephosphorylation assay. WT DnaK was heterologously expressed in E. coli with an N-terminal 6xHis-tag and purified. We also constructed and purified a non-phosphorylatable version of protein, DnaK Y601F, in which the phosphorylated tyrosine 601 was replaced by phenylalanine, thus removing the hydroxyl group required for phosphorylation. The mutation of Y601F did not affect the folding of DnaK, as demonstrated by very similar circular dichroism spectra of the DnaK WT and Y601F (Figure 2). We therefore concluded that the mutation is structure-neutral, and can be used to mimic the non-phosphorylated state of the protein. Pro-Q® Diamond phosphoprotein staining suggested that both DnaK and DnaK Y601F were to some extent phosphorylated during heterologous expression in E. coli (Figure 3A). This was confirmed by an independent method, Western blotting with an anti-phosphotyrosine antibody (Supplementary Figure 2). When incubated with PtkA and ATP, the phosphorylation signal increased both for DnaK and DnaK Y601F, and PtpZ was able to remove the phosphorylation signal (Figure 3A). This confirmed that DnaK can be phosphorylated by PtkA and dephosphorylated by PtpZ, but left some doubts as to the identity of the phosphorylated residue. To differentiate between the pre-existing phosphorylation of DnaK and its phosphorylation catalyzed by PtkA, we performed a phosphorylation assay based on incorporation of 32P from 32P-gamma-labeled ATP (Figure 3B). Here it was clear that phosphorylation of DnaK was more efficient than that of DnaK Y601F. However, some residual phosphorylation of DnaK Y601F occurred on residue(s) other than Y601, consistent with the results of Pro-Q® Diamond phosphoprotein staining. Using mass spectrometry, we identified another DnaK residue phosphorylated by PtkA in vitro: the tyrosine 573 (Supplementary Figure 3). We concluded that Y601 is the main residue phosphorylated by PtkA, and Y573 is the secondary phosphorylation site.


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)

Y601 of DnaK is phosphorylated by PtkA and dephosphorylated by PtpZ in vitro. Bands corresponding to phosphorylated DnaK and PtkA are indicated by arrows. (A) Phosphorylation and dephosphorylation assays by Pro-Q Diamond Phosphoprotein Gel Staining. In lane 1–5, reactions were performed with 1.2 μM PtkA, 1.2 μM TkmA, 3.6 μM DnaK WT, or Y601F, and incubated for 30 min. Presence of key proteins in the assays is indicated with ± above each lane. In lanes 6–8, 5 μM PtpZ was added into pre-incubated reaction containing 0.6 μM PtkA, 0.6 μM TkmA, and 3.6 μM DnaK WT. The incubation times are given above each lane. (B) Autoradiography images of the phosphorylation of DnaK WT (lanes 2–4) and Y601F (lanes 5–7) in the presence of PtkA, and dephosphorylating of DnaK WT (lanes 8–11) in the presence of PtpZ. Phosphorylation assay was performed with 1 μM PtkA, 1 μM TkmA, and 3 μM DnaK. PtkA was incubated alone in the lane 1. In dephosphorylation assay, 5 μM PtpZ was added into pre-incubated reaction containing 1 μM PtkA, 1 μM TkmA, and 3 μM DnaK WT. The incubation times are given above each lane.
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Related In: Results  -  Collection

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Figure 3: Y601 of DnaK is phosphorylated by PtkA and dephosphorylated by PtpZ in vitro. Bands corresponding to phosphorylated DnaK and PtkA are indicated by arrows. (A) Phosphorylation and dephosphorylation assays by Pro-Q Diamond Phosphoprotein Gel Staining. In lane 1–5, reactions were performed with 1.2 μM PtkA, 1.2 μM TkmA, 3.6 μM DnaK WT, or Y601F, and incubated for 30 min. Presence of key proteins in the assays is indicated with ± above each lane. In lanes 6–8, 5 μM PtpZ was added into pre-incubated reaction containing 0.6 μM PtkA, 0.6 μM TkmA, and 3.6 μM DnaK WT. The incubation times are given above each lane. (B) Autoradiography images of the phosphorylation of DnaK WT (lanes 2–4) and Y601F (lanes 5–7) in the presence of PtkA, and dephosphorylating of DnaK WT (lanes 8–11) in the presence of PtpZ. Phosphorylation assay was performed with 1 μM PtkA, 1 μM TkmA, and 3 μM DnaK. PtkA was incubated alone in the lane 1. In dephosphorylation assay, 5 μM PtpZ was added into pre-incubated reaction containing 1 μM PtkA, 1 μM TkmA, and 3 μM DnaK WT. The incubation times are given above each lane.
Mentions: Following the triple SILAC screening, we set out to explore the functional relevance of one differentially phosphorylated phosphotyrosine-site, Y601 of DnaK. Manual validation of the MS/MS spectrum of the phosphorylated DnaK peptide showed good coverage and annotation of fragment ions (Figure 1B). In order to assess whether DnaK can be phosphorylated by PtkA and dephosphorylated by PtpZ, we proceeded with an in vitro phosphorylation/dephosphorylation assay. WT DnaK was heterologously expressed in E. coli with an N-terminal 6xHis-tag and purified. We also constructed and purified a non-phosphorylatable version of protein, DnaK Y601F, in which the phosphorylated tyrosine 601 was replaced by phenylalanine, thus removing the hydroxyl group required for phosphorylation. The mutation of Y601F did not affect the folding of DnaK, as demonstrated by very similar circular dichroism spectra of the DnaK WT and Y601F (Figure 2). We therefore concluded that the mutation is structure-neutral, and can be used to mimic the non-phosphorylated state of the protein. Pro-Q® Diamond phosphoprotein staining suggested that both DnaK and DnaK Y601F were to some extent phosphorylated during heterologous expression in E. coli (Figure 3A). This was confirmed by an independent method, Western blotting with an anti-phosphotyrosine antibody (Supplementary Figure 2). When incubated with PtkA and ATP, the phosphorylation signal increased both for DnaK and DnaK Y601F, and PtpZ was able to remove the phosphorylation signal (Figure 3A). This confirmed that DnaK can be phosphorylated by PtkA and dephosphorylated by PtpZ, but left some doubts as to the identity of the phosphorylated residue. To differentiate between the pre-existing phosphorylation of DnaK and its phosphorylation catalyzed by PtkA, we performed a phosphorylation assay based on incorporation of 32P from 32P-gamma-labeled ATP (Figure 3B). Here it was clear that phosphorylation of DnaK was more efficient than that of DnaK Y601F. However, some residual phosphorylation of DnaK Y601F occurred on residue(s) other than Y601, consistent with the results of Pro-Q® Diamond phosphoprotein staining. Using mass spectrometry, we identified another DnaK residue phosphorylated by PtkA in vitro: the tyrosine 573 (Supplementary Figure 3). We concluded that Y601 is the main residue phosphorylated by PtkA, and Y573 is the secondary phosphorylation site.

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