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

Lei Shi (Institutionen för biologi och bioteknik, Systembiologi) ; V. Ravikumar ; Abderahmane Derouiche (Institutionen för biologi och bioteknik, Systembiologi) ; B. Macek ; Ivan Mijakovic (Institutionen för biologi och bioteknik, Systembiologi)
Frontiers in Microbiology (1664-302X). Vol. 7 (2016),
[Artikel, refereegranskad vetenskaplig]

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 Delta ptkA strain and increased in the Delta ptpZ 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 refolding 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.

Nyckelord: quantitative phosphoproteomics, bacterial protein-tyrosine kinases, molecular chaperones, protein

Denna post skapades 2016-05-27. Senast ändrad 2016-05-30.
CPL Pubid: 237004


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Institutionen för biologi och bioteknik, Systembiologi


Biokemi och molekylärbiologi

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