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Interaction of bacterial fatty-acid-displaced regulators with DNA is interrupted by tyrosine phosphorylation in the helix-turn-helix domain.

Derouiche A, Bidnenko V, Grenha R, Pigonneau N, Ventroux M, Franz-Wachtel M, Nessler S, Noirot-Gros MF, Mijakovic I - Nucleic Acids Res. (2013)

Bottom Line: FatR was found to interact in a two-hybrid assay with TkmA, an activator of the protein-tyrosine kinase PtkA.Structural modelling reveals that the hydroxyl group of tyrosine 45 interacts with DNA, and we show that this phosphorylation reduces FatR DNA binding capacity.This indicates that phosphorylation of tyrosine 45 may be a general mechanism of switching off bacterial fatty-acid-displaced regulators.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1319 Micalis, 78350 Jouy-en-Josas, France, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Université Paris-Sud 11, 91405 Orsay, France, Proteome Center Tübingen, University of Tübingen, 72076 Tübingen, Germany and Department of Chemical and Biological Engineering, Chalmers University of Technology, 41296 Gothenburg, Sweden.

ABSTRACT
Bacteria possess transcription regulators (of the TetR family) specifically dedicated to repressing genes for cytochrome P450, involved in oxidation of polyunsaturated fatty acids. Interaction of these repressors with operator sequences is disrupted in the presence of fatty acids, and they are therefore known as fatty-acid-displaced regulators. Here, we describe a novel mechanism of inactivating the interaction of these proteins with DNA, illustrated by the example of Bacillus subtilis regulator FatR. FatR was found to interact in a two-hybrid assay with TkmA, an activator of the protein-tyrosine kinase PtkA. We show that FatR is phosphorylated specifically at the residue tyrosine 45 in its helix-turn-helix domain by the kinase PtkA. Structural modelling reveals that the hydroxyl group of tyrosine 45 interacts with DNA, and we show that this phosphorylation reduces FatR DNA binding capacity. Point mutants mimicking phosphorylation of FatR in vivo lead to a strong derepression of the fatR operon, indicating that this regulatory mechanism works independently of derepression by polyunsaturated fatty acids. Tyrosine 45 is a highly conserved residue, and PtkA from B. subtilis can phosphorylate FatR homologues from other bacteria. This indicates that phosphorylation of tyrosine 45 may be a general mechanism of switching off bacterial fatty-acid-displaced regulators.

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Related in: MedlinePlus

PtkA phosphorylates FatR at the residue tyrosine 45. (A) Specific interaction of the C-terminal part of FatR (residues 101–194) with TkmA in the yeast two-hybrid assay. Gene fatR 101–194 was cloned in the plasmids pGAD as a translational fusion with activating domain of the gal4 regulator, and tkmA was cloned in pGBDU, in fusion with the binding domain of gal4. Two clones for each construct were tested, designated (1) and (2). Vectors without fatR 101–194 and tkmA were used as negative controls. Eight days after the drop of yeast cells on the selective (−)LUH SD medium, the development of colonies was observed for tested strains expressing interacting proteins. (B) In vitro phosphorylation assay of FatR. Presence of purified proteins is indicated above each reaction lane. All reactions contained [γ-32P] ATP, MgCl2 and Tris-HCl, pH 7.5: concentrations and reaction conditions are given in Materials and Methods section. In the left, PtkA and FatR are from B. subtilis, and in the right, they are from B. megaterium. TkmA is from B. subtilis in all reactions. (C) After in vitro phosphorylation of B. subtilis FatR by PtkA, the sample was digested in solution with trypsine, and phosphopeptides were enriched by titanium dioxide chromatography and subjected to mass spectrometry. The spectrum shows the fragmentation pattern of the FatR phosphopeptide AHVGTGTIY(ph)R phosphorylated at the tyrosine 45.
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gkt709-F1: PtkA phosphorylates FatR at the residue tyrosine 45. (A) Specific interaction of the C-terminal part of FatR (residues 101–194) with TkmA in the yeast two-hybrid assay. Gene fatR 101–194 was cloned in the plasmids pGAD as a translational fusion with activating domain of the gal4 regulator, and tkmA was cloned in pGBDU, in fusion with the binding domain of gal4. Two clones for each construct were tested, designated (1) and (2). Vectors without fatR 101–194 and tkmA were used as negative controls. Eight days after the drop of yeast cells on the selective (−)LUH SD medium, the development of colonies was observed for tested strains expressing interacting proteins. (B) In vitro phosphorylation assay of FatR. Presence of purified proteins is indicated above each reaction lane. All reactions contained [γ-32P] ATP, MgCl2 and Tris-HCl, pH 7.5: concentrations and reaction conditions are given in Materials and Methods section. In the left, PtkA and FatR are from B. subtilis, and in the right, they are from B. megaterium. TkmA is from B. subtilis in all reactions. (C) After in vitro phosphorylation of B. subtilis FatR by PtkA, the sample was digested in solution with trypsine, and phosphopeptides were enriched by titanium dioxide chromatography and subjected to mass spectrometry. The spectrum shows the fragmentation pattern of the FatR phosphopeptide AHVGTGTIY(ph)R phosphorylated at the tyrosine 45.

Mentions: To search for new interactants of PtkA and TkmA, we performed a two-hybrid screen against the B. subtilis genomic library (27). Among others (data not shown), we detected a specific interaction between TkmA and the C-terminal domain (residues 101–194) of the fatty-acid-displaced regulator FatR. The results of specificity test for this interaction are presented in Figure 1A. Development of colonies was observed only for strains containing the plasmid pGAD, with the activating domain of the gal4 gene fused to the tkmA and the pGBDU plasmid with the binding domain of the gal4 fused to the C-terminal fragment of fatR. Negative controls with empty plasmids confirm that the simultaneous presence of FatR and TkmA was required for growth. We concluded that TkmA and FatR interact in the yeast two-hybrid assay, mimicking the in vivo conditions. Because TkmA is known to activate the BY-kinase PtkA and promote substrate phosphorylation presumably in a ternary complex (TkmA-PtkA-substrate) (7), we tested whether PtkA would be able to phosphorylate FatR in the presence of TkmA. For this, we set up an in vitro phosphorylation assay, using purified 6xHis-tagged proteins. Proteins were mixed with [γ-32P] ATP, and incorporation of radioactive phosphate was revealed by autoradiography. As can be observed in Figure 1B (left), FatR was not able to autophosphorylate in the presence of [γ-32P] ATP. In the absence of TkmA, PtkA could not phosphorylate FatR. However, a radioactive signal associated to FatR appeared when both PtkA and TkmA were present in the reaction, indicating that FatR could be phosphorylated by PtkA. Since the FatR homologue from B. megaterium (Bm3R1) was the first characterized member of this regulator family, we also examined whether it can be phosphorylated by its cognate kinase (product of the gene bmQ-1130 identified by BLAST). We purified the B. megaterium FatR and PtkA proteins and performed the same type of in vitro phosphorylation assay. The result was identical to that obtained with B. subtilis proteins, and B. megaterium FatR was phosphorylated by the cognate PtkA in the presence of B. subtilis TkmA (Figure 1B, right). To determine the exact site of B. subtilis FatR phosphorylation, we performed the phosphorylation reaction in exactly the same conditions, but without radioactive ATP. We then attempted to detect the phosphorylation site on FatR using mass spectrometry analysis. Phosphorylation was unambiguously detected at the residue tyrosine 45, with the probability score of 0.976 (Figure 1C). We thus concluded that PtkA catalyzes a specific phosphorylation of FatR at the residue tyrosine 45.Figure 1.


Interaction of bacterial fatty-acid-displaced regulators with DNA is interrupted by tyrosine phosphorylation in the helix-turn-helix domain.

Derouiche A, Bidnenko V, Grenha R, Pigonneau N, Ventroux M, Franz-Wachtel M, Nessler S, Noirot-Gros MF, Mijakovic I - Nucleic Acids Res. (2013)

PtkA phosphorylates FatR at the residue tyrosine 45. (A) Specific interaction of the C-terminal part of FatR (residues 101–194) with TkmA in the yeast two-hybrid assay. Gene fatR 101–194 was cloned in the plasmids pGAD as a translational fusion with activating domain of the gal4 regulator, and tkmA was cloned in pGBDU, in fusion with the binding domain of gal4. Two clones for each construct were tested, designated (1) and (2). Vectors without fatR 101–194 and tkmA were used as negative controls. Eight days after the drop of yeast cells on the selective (−)LUH SD medium, the development of colonies was observed for tested strains expressing interacting proteins. (B) In vitro phosphorylation assay of FatR. Presence of purified proteins is indicated above each reaction lane. All reactions contained [γ-32P] ATP, MgCl2 and Tris-HCl, pH 7.5: concentrations and reaction conditions are given in Materials and Methods section. In the left, PtkA and FatR are from B. subtilis, and in the right, they are from B. megaterium. TkmA is from B. subtilis in all reactions. (C) After in vitro phosphorylation of B. subtilis FatR by PtkA, the sample was digested in solution with trypsine, and phosphopeptides were enriched by titanium dioxide chromatography and subjected to mass spectrometry. The spectrum shows the fragmentation pattern of the FatR phosphopeptide AHVGTGTIY(ph)R phosphorylated at the tyrosine 45.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814354&req=5

gkt709-F1: PtkA phosphorylates FatR at the residue tyrosine 45. (A) Specific interaction of the C-terminal part of FatR (residues 101–194) with TkmA in the yeast two-hybrid assay. Gene fatR 101–194 was cloned in the plasmids pGAD as a translational fusion with activating domain of the gal4 regulator, and tkmA was cloned in pGBDU, in fusion with the binding domain of gal4. Two clones for each construct were tested, designated (1) and (2). Vectors without fatR 101–194 and tkmA were used as negative controls. Eight days after the drop of yeast cells on the selective (−)LUH SD medium, the development of colonies was observed for tested strains expressing interacting proteins. (B) In vitro phosphorylation assay of FatR. Presence of purified proteins is indicated above each reaction lane. All reactions contained [γ-32P] ATP, MgCl2 and Tris-HCl, pH 7.5: concentrations and reaction conditions are given in Materials and Methods section. In the left, PtkA and FatR are from B. subtilis, and in the right, they are from B. megaterium. TkmA is from B. subtilis in all reactions. (C) After in vitro phosphorylation of B. subtilis FatR by PtkA, the sample was digested in solution with trypsine, and phosphopeptides were enriched by titanium dioxide chromatography and subjected to mass spectrometry. The spectrum shows the fragmentation pattern of the FatR phosphopeptide AHVGTGTIY(ph)R phosphorylated at the tyrosine 45.
Mentions: To search for new interactants of PtkA and TkmA, we performed a two-hybrid screen against the B. subtilis genomic library (27). Among others (data not shown), we detected a specific interaction between TkmA and the C-terminal domain (residues 101–194) of the fatty-acid-displaced regulator FatR. The results of specificity test for this interaction are presented in Figure 1A. Development of colonies was observed only for strains containing the plasmid pGAD, with the activating domain of the gal4 gene fused to the tkmA and the pGBDU plasmid with the binding domain of the gal4 fused to the C-terminal fragment of fatR. Negative controls with empty plasmids confirm that the simultaneous presence of FatR and TkmA was required for growth. We concluded that TkmA and FatR interact in the yeast two-hybrid assay, mimicking the in vivo conditions. Because TkmA is known to activate the BY-kinase PtkA and promote substrate phosphorylation presumably in a ternary complex (TkmA-PtkA-substrate) (7), we tested whether PtkA would be able to phosphorylate FatR in the presence of TkmA. For this, we set up an in vitro phosphorylation assay, using purified 6xHis-tagged proteins. Proteins were mixed with [γ-32P] ATP, and incorporation of radioactive phosphate was revealed by autoradiography. As can be observed in Figure 1B (left), FatR was not able to autophosphorylate in the presence of [γ-32P] ATP. In the absence of TkmA, PtkA could not phosphorylate FatR. However, a radioactive signal associated to FatR appeared when both PtkA and TkmA were present in the reaction, indicating that FatR could be phosphorylated by PtkA. Since the FatR homologue from B. megaterium (Bm3R1) was the first characterized member of this regulator family, we also examined whether it can be phosphorylated by its cognate kinase (product of the gene bmQ-1130 identified by BLAST). We purified the B. megaterium FatR and PtkA proteins and performed the same type of in vitro phosphorylation assay. The result was identical to that obtained with B. subtilis proteins, and B. megaterium FatR was phosphorylated by the cognate PtkA in the presence of B. subtilis TkmA (Figure 1B, right). To determine the exact site of B. subtilis FatR phosphorylation, we performed the phosphorylation reaction in exactly the same conditions, but without radioactive ATP. We then attempted to detect the phosphorylation site on FatR using mass spectrometry analysis. Phosphorylation was unambiguously detected at the residue tyrosine 45, with the probability score of 0.976 (Figure 1C). We thus concluded that PtkA catalyzes a specific phosphorylation of FatR at the residue tyrosine 45.Figure 1.

Bottom Line: FatR was found to interact in a two-hybrid assay with TkmA, an activator of the protein-tyrosine kinase PtkA.Structural modelling reveals that the hydroxyl group of tyrosine 45 interacts with DNA, and we show that this phosphorylation reduces FatR DNA binding capacity.This indicates that phosphorylation of tyrosine 45 may be a general mechanism of switching off bacterial fatty-acid-displaced regulators.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR1319 Micalis, 78350 Jouy-en-Josas, France, Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Université Paris-Sud 11, 91405 Orsay, France, Proteome Center Tübingen, University of Tübingen, 72076 Tübingen, Germany and Department of Chemical and Biological Engineering, Chalmers University of Technology, 41296 Gothenburg, Sweden.

ABSTRACT
Bacteria possess transcription regulators (of the TetR family) specifically dedicated to repressing genes for cytochrome P450, involved in oxidation of polyunsaturated fatty acids. Interaction of these repressors with operator sequences is disrupted in the presence of fatty acids, and they are therefore known as fatty-acid-displaced regulators. Here, we describe a novel mechanism of inactivating the interaction of these proteins with DNA, illustrated by the example of Bacillus subtilis regulator FatR. FatR was found to interact in a two-hybrid assay with TkmA, an activator of the protein-tyrosine kinase PtkA. We show that FatR is phosphorylated specifically at the residue tyrosine 45 in its helix-turn-helix domain by the kinase PtkA. Structural modelling reveals that the hydroxyl group of tyrosine 45 interacts with DNA, and we show that this phosphorylation reduces FatR DNA binding capacity. Point mutants mimicking phosphorylation of FatR in vivo lead to a strong derepression of the fatR operon, indicating that this regulatory mechanism works independently of derepression by polyunsaturated fatty acids. Tyrosine 45 is a highly conserved residue, and PtkA from B. subtilis can phosphorylate FatR homologues from other bacteria. This indicates that phosphorylation of tyrosine 45 may be a general mechanism of switching off bacterial fatty-acid-displaced regulators.

Show MeSH
Related in: MedlinePlus