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Cycling of Etk and Etp phosphorylation states is involved in formation of group 4 capsule by Escherichia coli.

Nadler C, Koby S, Peleg A, Johnson AC, Suddala KC, Sathiyamoorthy K, Smith BE, Saper MA, Rosenshine I - PLoS ONE (2012)

Bottom Line: We show that Etp dephosphorylates Etk in vivo, and mutations rendering Etk or Etp catalytically inactive result in loss of group 4 capsule production, supporting the notion that cyclic phosphorylation and dephosphorylation of Etk is required for capsule formation.Although EtpY121E and EtpY121A still supported capsule formation, EtpY121F failed to do so.These results suggest that cycles of phosphorylation and dephosphorylation of Etp, as well as Etk, are involved in the formation of group 4 capsule, providing an additional regulatory layer to the complex control of capsule production.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT
Capsules frequently play a key role in bacterial interactions with their environment. Escherichia coli capsules were categorized as groups 1 through 4, each produced by a distinct mechanism. Etk and Etp are members of protein families required for the production of group 1 and group 4 capsules. These members function as a protein tyrosine kinase and protein tyrosine phosphatase, respectively. We show that Etp dephosphorylates Etk in vivo, and mutations rendering Etk or Etp catalytically inactive result in loss of group 4 capsule production, supporting the notion that cyclic phosphorylation and dephosphorylation of Etk is required for capsule formation. Notably, Etp also becomes tyrosine phosphorylated in vivo and catalyzes rapid auto-dephosphorylation. Further analysis identified Tyr121 as the phosphorylated residue of Etp. Etp containing Phe, Glu or Ala in place of Tyr121 retained phosphatase activity and catalyzed dephosphorylation of Etp and Etk. Although EtpY121E and EtpY121A still supported capsule formation, EtpY121F failed to do so. These results suggest that cycles of phosphorylation and dephosphorylation of Etp, as well as Etk, are involved in the formation of group 4 capsule, providing an additional regulatory layer to the complex control of capsule production.

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Related in: MedlinePlus

ESI tandem mass spectrometry confirms the presence and location of the phosphorylated tyrosine.The ESI-MS/MS fragmentation spectrum of Etp peptide GKTMLFGQWLEQKEIPAP(pY)RK (residues 113–123, pY is the phosphorylated tyrosine). The parent peptide was a +4 H ion with mass 2599.29 amu (−0.0134 amu from predicted size). The schematic at the top of the figure shows the identified y fragments referred to in the spectra. All of the y fragments include the peptide's C-terminus and have masses consistent with the phosphorylated Y121 (pY) residue. For example, the m/z for fragments y3 and y2 are 546.2 and 303.2, respectively. The difference 243 is the exact mass of a phosphotyrosyl residue. The labeled b fragments originate at the N-terminus of the peptide but do not include the pY residue.
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pone-0037984-g004: ESI tandem mass spectrometry confirms the presence and location of the phosphorylated tyrosine.The ESI-MS/MS fragmentation spectrum of Etp peptide GKTMLFGQWLEQKEIPAP(pY)RK (residues 113–123, pY is the phosphorylated tyrosine). The parent peptide was a +4 H ion with mass 2599.29 amu (−0.0134 amu from predicted size). The schematic at the top of the figure shows the identified y fragments referred to in the spectra. All of the y fragments include the peptide's C-terminus and have masses consistent with the phosphorylated Y121 (pY) residue. For example, the m/z for fragments y3 and y2 are 546.2 and 303.2, respectively. The difference 243 is the exact mass of a phosphotyrosyl residue. The labeled b fragments originate at the N-terminus of the peptide but do not include the pY residue.

Mentions: We noticed that Etp phosphorylation was not restricted to EPEC and was also observed in other E. coli strains. We took advantage of this observation to further examine the finding that Tyr121 was phosphorylated. EtpD119A was expressed from pAJ0046 in E. coli BL21(DE3) and purified. Western blotting with anti-phosphotyrosine antibody 4G10 (Millipore/Upstate) confirmed that the protein was hyper-phosphorylated when compared to wild type Etp (data not shown). The purified protein was analyzed by electrospray tandem mass spectrometry (LC-MS/MS) by MS Bioworks, LLC (Ann Arbor MI). The size of peptides that included Tyr121 as well as their fragmentation patterns conclusively showed the presence of a phosphorylated tyrosine (+80 a.m.u.) corresponding to position 121 of the native protein (Fig. 4). This was observed in 31 of 64 spectra of peptides containing Tyr121. Fig. 4 is a typical spectrum showing C-terminal fragments before (y3) and after (y2) the phosphorylated Tyr121. No tyrosine phosphorylation was observed elsewhere in the protein.


Cycling of Etk and Etp phosphorylation states is involved in formation of group 4 capsule by Escherichia coli.

Nadler C, Koby S, Peleg A, Johnson AC, Suddala KC, Sathiyamoorthy K, Smith BE, Saper MA, Rosenshine I - PLoS ONE (2012)

ESI tandem mass spectrometry confirms the presence and location of the phosphorylated tyrosine.The ESI-MS/MS fragmentation spectrum of Etp peptide GKTMLFGQWLEQKEIPAP(pY)RK (residues 113–123, pY is the phosphorylated tyrosine). The parent peptide was a +4 H ion with mass 2599.29 amu (−0.0134 amu from predicted size). The schematic at the top of the figure shows the identified y fragments referred to in the spectra. All of the y fragments include the peptide's C-terminus and have masses consistent with the phosphorylated Y121 (pY) residue. For example, the m/z for fragments y3 and y2 are 546.2 and 303.2, respectively. The difference 243 is the exact mass of a phosphotyrosyl residue. The labeled b fragments originate at the N-terminus of the peptide but do not include the pY residue.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366997&req=5

pone-0037984-g004: ESI tandem mass spectrometry confirms the presence and location of the phosphorylated tyrosine.The ESI-MS/MS fragmentation spectrum of Etp peptide GKTMLFGQWLEQKEIPAP(pY)RK (residues 113–123, pY is the phosphorylated tyrosine). The parent peptide was a +4 H ion with mass 2599.29 amu (−0.0134 amu from predicted size). The schematic at the top of the figure shows the identified y fragments referred to in the spectra. All of the y fragments include the peptide's C-terminus and have masses consistent with the phosphorylated Y121 (pY) residue. For example, the m/z for fragments y3 and y2 are 546.2 and 303.2, respectively. The difference 243 is the exact mass of a phosphotyrosyl residue. The labeled b fragments originate at the N-terminus of the peptide but do not include the pY residue.
Mentions: We noticed that Etp phosphorylation was not restricted to EPEC and was also observed in other E. coli strains. We took advantage of this observation to further examine the finding that Tyr121 was phosphorylated. EtpD119A was expressed from pAJ0046 in E. coli BL21(DE3) and purified. Western blotting with anti-phosphotyrosine antibody 4G10 (Millipore/Upstate) confirmed that the protein was hyper-phosphorylated when compared to wild type Etp (data not shown). The purified protein was analyzed by electrospray tandem mass spectrometry (LC-MS/MS) by MS Bioworks, LLC (Ann Arbor MI). The size of peptides that included Tyr121 as well as their fragmentation patterns conclusively showed the presence of a phosphorylated tyrosine (+80 a.m.u.) corresponding to position 121 of the native protein (Fig. 4). This was observed in 31 of 64 spectra of peptides containing Tyr121. Fig. 4 is a typical spectrum showing C-terminal fragments before (y3) and after (y2) the phosphorylated Tyr121. No tyrosine phosphorylation was observed elsewhere in the protein.

Bottom Line: We show that Etp dephosphorylates Etk in vivo, and mutations rendering Etk or Etp catalytically inactive result in loss of group 4 capsule production, supporting the notion that cyclic phosphorylation and dephosphorylation of Etk is required for capsule formation.Although EtpY121E and EtpY121A still supported capsule formation, EtpY121F failed to do so.These results suggest that cycles of phosphorylation and dephosphorylation of Etp, as well as Etk, are involved in the formation of group 4 capsule, providing an additional regulatory layer to the complex control of capsule production.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, The Hebrew University-Hadassah Medical School, Jerusalem, Israel.

ABSTRACT
Capsules frequently play a key role in bacterial interactions with their environment. Escherichia coli capsules were categorized as groups 1 through 4, each produced by a distinct mechanism. Etk and Etp are members of protein families required for the production of group 1 and group 4 capsules. These members function as a protein tyrosine kinase and protein tyrosine phosphatase, respectively. We show that Etp dephosphorylates Etk in vivo, and mutations rendering Etk or Etp catalytically inactive result in loss of group 4 capsule production, supporting the notion that cyclic phosphorylation and dephosphorylation of Etk is required for capsule formation. Notably, Etp also becomes tyrosine phosphorylated in vivo and catalyzes rapid auto-dephosphorylation. Further analysis identified Tyr121 as the phosphorylated residue of Etp. Etp containing Phe, Glu or Ala in place of Tyr121 retained phosphatase activity and catalyzed dephosphorylation of Etp and Etk. Although EtpY121E and EtpY121A still supported capsule formation, EtpY121F failed to do so. These results suggest that cycles of phosphorylation and dephosphorylation of Etp, as well as Etk, are involved in the formation of group 4 capsule, providing an additional regulatory layer to the complex control of capsule production.

Show MeSH
Related in: MedlinePlus