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SpvC is a Salmonella effector with phosphothreonine lyase activity on host mitogen-activated protein kinases.

Mazurkiewicz P, Thomas J, Thompson JA, Liu M, Arbibe L, Sansonetti P, Holden DW - Mol. Microbiol. (2008)

Bottom Line: SpvC is encoded by the Salmonella virulence plasmid.SpvC can be secreted in vitro by either the SPI-1 or SPI-2 type III secretion systems.Using antibodies specific to phospho-amino acids and mass spectrometry we demonstrate that SpvC has phosphothreonine lyase activity on full-length phospho-Erk (pErk) and a synthetic 13-amino-acid phospho-peptide containing the TXY motif.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK.

ABSTRACT
SpvC is encoded by the Salmonella virulence plasmid. We have investigated the biochemical function of SpvC and the mechanism by which it is secreted by bacteria and translocated into infected macrophages. We constructed a strain carrying a deletion in spvC and showed that the strain is attenuated for systemic virulence in mice. SpvC can be secreted in vitro by either the SPI-1 or SPI-2 type III secretion systems. Cell biological and genetic experiments showed that translocation of the protein into the cytosol of macrophages by intracellular bacteria is dependent on the SPI-2 T3SS. Using antibodies specific to phospho-amino acids and mass spectrometry we demonstrate that SpvC has phosphothreonine lyase activity on full-length phospho-Erk (pErk) and a synthetic 13-amino-acid phospho-peptide containing the TXY motif. A Salmonella strain expressing spvC from a plasmid downregulated cytokine release from infected cells.

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Purified GST–SpvC removes phosphate group(s) from pErk and pJNK in vitro. Phosphorylation status of Erk/JNK was determined using an antibody specific for double (threonine and tyrosine) phosphorylated MAP kinases (upper panels in A, B and C). Antibodies detecting MAP kinases regardless of their phosphorylation state were used to check the amounts of pErk/pJNK used (lower panels in A, B and C). A. One hundred nanograms of pErk were incubated with 30 U (1 μg) of MAP-kinase phosphatase 1 (MKP1), 400 ng of GST–SpvC, 400 ng of GST or left untreated as a positive control for anti-pErk antibody. B. One hundred nanograms of pJNK were incubated with 30 U MKP1, 400 ng of GST–SpvC or left untreated as a positive control for anti-pJNK antibody. C. Effect of orthovanadate on the activity of MKP1 and GST–SpvC. Two hundred nanograms of pErk were incubated with 1 μg of MKP1, 200 ng of GST–SpvC and indicated concentrations of sodium orthovanadate.
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fig05: Purified GST–SpvC removes phosphate group(s) from pErk and pJNK in vitro. Phosphorylation status of Erk/JNK was determined using an antibody specific for double (threonine and tyrosine) phosphorylated MAP kinases (upper panels in A, B and C). Antibodies detecting MAP kinases regardless of their phosphorylation state were used to check the amounts of pErk/pJNK used (lower panels in A, B and C). A. One hundred nanograms of pErk were incubated with 30 U (1 μg) of MAP-kinase phosphatase 1 (MKP1), 400 ng of GST–SpvC, 400 ng of GST or left untreated as a positive control for anti-pErk antibody. B. One hundred nanograms of pJNK were incubated with 30 U MKP1, 400 ng of GST–SpvC or left untreated as a positive control for anti-pJNK antibody. C. Effect of orthovanadate on the activity of MKP1 and GST–SpvC. Two hundred nanograms of pErk were incubated with 1 μg of MKP1, 200 ng of GST–SpvC and indicated concentrations of sodium orthovanadate.

Mentions: To establish if SpvC is sufficient for Erk inactivation we purified GST–SpvC (Fig. S1), GST and SpvC-His and used these in in vitro dephosphorylation assays with purified, phosphorylated MAP kinases. A dual specificity MAP kinase phosphatase 1 (MKP1) was used as a control for MAP kinase dephosphorylation. Immunoblotting analysis revealed that MKP1 and GST–SpvC can remove phosphate groups from pErk (Fig. 5A). Similar results were obtained when pJNK was incubated with MKP1 and GST–SpvC (Fig. 5B). Next we tested the sensitivity of SpvC to vanadate, a tyrosine phosphatase inhibitor. pErk was incubated with GST–SpvC in the presence or absence of vanadate. As a control, MKP1 (which is susceptible to vanadate inhibition) was used (Charles et al., 1993). As expected, dephosphorylation of pErk by MKP1 was inhibited by addition of vanadate, but the activity of SpvC was not (Fig. 5C), indicating that SpvC might not be a typical dual specificity phosphatase.


SpvC is a Salmonella effector with phosphothreonine lyase activity on host mitogen-activated protein kinases.

Mazurkiewicz P, Thomas J, Thompson JA, Liu M, Arbibe L, Sansonetti P, Holden DW - Mol. Microbiol. (2008)

Purified GST–SpvC removes phosphate group(s) from pErk and pJNK in vitro. Phosphorylation status of Erk/JNK was determined using an antibody specific for double (threonine and tyrosine) phosphorylated MAP kinases (upper panels in A, B and C). Antibodies detecting MAP kinases regardless of their phosphorylation state were used to check the amounts of pErk/pJNK used (lower panels in A, B and C). A. One hundred nanograms of pErk were incubated with 30 U (1 μg) of MAP-kinase phosphatase 1 (MKP1), 400 ng of GST–SpvC, 400 ng of GST or left untreated as a positive control for anti-pErk antibody. B. One hundred nanograms of pJNK were incubated with 30 U MKP1, 400 ng of GST–SpvC or left untreated as a positive control for anti-pJNK antibody. C. Effect of orthovanadate on the activity of MKP1 and GST–SpvC. Two hundred nanograms of pErk were incubated with 1 μg of MKP1, 200 ng of GST–SpvC and indicated concentrations of sodium orthovanadate.
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getmorefigures.php?uid=PMC2268955&req=5

fig05: Purified GST–SpvC removes phosphate group(s) from pErk and pJNK in vitro. Phosphorylation status of Erk/JNK was determined using an antibody specific for double (threonine and tyrosine) phosphorylated MAP kinases (upper panels in A, B and C). Antibodies detecting MAP kinases regardless of their phosphorylation state were used to check the amounts of pErk/pJNK used (lower panels in A, B and C). A. One hundred nanograms of pErk were incubated with 30 U (1 μg) of MAP-kinase phosphatase 1 (MKP1), 400 ng of GST–SpvC, 400 ng of GST or left untreated as a positive control for anti-pErk antibody. B. One hundred nanograms of pJNK were incubated with 30 U MKP1, 400 ng of GST–SpvC or left untreated as a positive control for anti-pJNK antibody. C. Effect of orthovanadate on the activity of MKP1 and GST–SpvC. Two hundred nanograms of pErk were incubated with 1 μg of MKP1, 200 ng of GST–SpvC and indicated concentrations of sodium orthovanadate.
Mentions: To establish if SpvC is sufficient for Erk inactivation we purified GST–SpvC (Fig. S1), GST and SpvC-His and used these in in vitro dephosphorylation assays with purified, phosphorylated MAP kinases. A dual specificity MAP kinase phosphatase 1 (MKP1) was used as a control for MAP kinase dephosphorylation. Immunoblotting analysis revealed that MKP1 and GST–SpvC can remove phosphate groups from pErk (Fig. 5A). Similar results were obtained when pJNK was incubated with MKP1 and GST–SpvC (Fig. 5B). Next we tested the sensitivity of SpvC to vanadate, a tyrosine phosphatase inhibitor. pErk was incubated with GST–SpvC in the presence or absence of vanadate. As a control, MKP1 (which is susceptible to vanadate inhibition) was used (Charles et al., 1993). As expected, dephosphorylation of pErk by MKP1 was inhibited by addition of vanadate, but the activity of SpvC was not (Fig. 5C), indicating that SpvC might not be a typical dual specificity phosphatase.

Bottom Line: SpvC is encoded by the Salmonella virulence plasmid.SpvC can be secreted in vitro by either the SPI-1 or SPI-2 type III secretion systems.Using antibodies specific to phospho-amino acids and mass spectrometry we demonstrate that SpvC has phosphothreonine lyase activity on full-length phospho-Erk (pErk) and a synthetic 13-amino-acid phospho-peptide containing the TXY motif.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK.

ABSTRACT
SpvC is encoded by the Salmonella virulence plasmid. We have investigated the biochemical function of SpvC and the mechanism by which it is secreted by bacteria and translocated into infected macrophages. We constructed a strain carrying a deletion in spvC and showed that the strain is attenuated for systemic virulence in mice. SpvC can be secreted in vitro by either the SPI-1 or SPI-2 type III secretion systems. Cell biological and genetic experiments showed that translocation of the protein into the cytosol of macrophages by intracellular bacteria is dependent on the SPI-2 T3SS. Using antibodies specific to phospho-amino acids and mass spectrometry we demonstrate that SpvC has phosphothreonine lyase activity on full-length phospho-Erk (pErk) and a synthetic 13-amino-acid phospho-peptide containing the TXY motif. A Salmonella strain expressing spvC from a plasmid downregulated cytokine release from infected cells.

Show MeSH
Related in: MedlinePlus