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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

Etp promotes Etk dephosphorylation and is tyrosine phosphorylated in vivo.EPEC strains transformed, or not transformed, with plasmid pAP406 (p6His-Etp) expressing 6His-Etp, were grown overnight in LB at 20°C without shaking. The bacteria were harvested, and the extracted proteins were separated for Western blot analysis with anti-Etk, anti-phosphotyrosine (anti-PY), and anti-6His antibodies. The corresponding antibody is indicated above each panel. Also indicated are the location of Etk and 6His-Etp and the molecular size markers (at the right). Whether a given strain contained the pEtp plasmid is indicated below each lane.
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pone-0037984-g002: Etp promotes Etk dephosphorylation and is tyrosine phosphorylated in vivo.EPEC strains transformed, or not transformed, with plasmid pAP406 (p6His-Etp) expressing 6His-Etp, were grown overnight in LB at 20°C without shaking. The bacteria were harvested, and the extracted proteins were separated for Western blot analysis with anti-Etk, anti-phosphotyrosine (anti-PY), and anti-6His antibodies. The corresponding antibody is indicated above each panel. Also indicated are the location of Etk and 6His-Etp and the molecular size markers (at the right). Whether a given strain contained the pEtp plasmid is indicated below each lane.

Mentions: To test if Etp can dephosphorylate Etk in vivo, we transformed EPEC with a plasmid encoding 6His-Etp (pAP406) and expressed the recombinant Etp in LB at 20°C without shaking (conditions that enhanced Etp solubility). As control, we used wild type EPEC that did not express the recombinant Etp. Proteins were extracted from the two strains and analyzed by Western blot with anti-Etk, anti-6His and anti-phosphotyrosine (anti-PY) antibodies. The results show a clear decrease in tyrosine phosphorylated Etk upon Etp overexpression (Fig. 2), suggesting that Etp dephosphorylates Etk in vivo. Intriguingly, we noticed that the anti-PY antibody also reacted with the recombinant Etp (Fig. 2), indicating that some of the Etp was tyrosine phosphorylated. We also noted that the blotted Etp regained some of its acid-phosphatase activity during the blot development to catalyze autodephosphorylation (Fig. S1). To reduce the autodephosphorylation activity, blots were developed at pH 8 to inhibit Etp (Fig. S2). To exclude the possibility that the anti-PY antibody recognized Etp by nonspecific cross-reaction, we included the PY competitive inhibitor phenylphosphate that abolished the antibody interaction with Etk (Fig. S3). Taken together, our results show that Etp becomes tyrosine phosphorylated in vivo and that it catalyzes autodephosphorylation.


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)

Etp promotes Etk dephosphorylation and is tyrosine phosphorylated in vivo.EPEC strains transformed, or not transformed, with plasmid pAP406 (p6His-Etp) expressing 6His-Etp, were grown overnight in LB at 20°C without shaking. The bacteria were harvested, and the extracted proteins were separated for Western blot analysis with anti-Etk, anti-phosphotyrosine (anti-PY), and anti-6His antibodies. The corresponding antibody is indicated above each panel. Also indicated are the location of Etk and 6His-Etp and the molecular size markers (at the right). Whether a given strain contained the pEtp plasmid is indicated below each lane.
© Copyright Policy
Related In: Results  -  Collection

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pone-0037984-g002: Etp promotes Etk dephosphorylation and is tyrosine phosphorylated in vivo.EPEC strains transformed, or not transformed, with plasmid pAP406 (p6His-Etp) expressing 6His-Etp, were grown overnight in LB at 20°C without shaking. The bacteria were harvested, and the extracted proteins were separated for Western blot analysis with anti-Etk, anti-phosphotyrosine (anti-PY), and anti-6His antibodies. The corresponding antibody is indicated above each panel. Also indicated are the location of Etk and 6His-Etp and the molecular size markers (at the right). Whether a given strain contained the pEtp plasmid is indicated below each lane.
Mentions: To test if Etp can dephosphorylate Etk in vivo, we transformed EPEC with a plasmid encoding 6His-Etp (pAP406) and expressed the recombinant Etp in LB at 20°C without shaking (conditions that enhanced Etp solubility). As control, we used wild type EPEC that did not express the recombinant Etp. Proteins were extracted from the two strains and analyzed by Western blot with anti-Etk, anti-6His and anti-phosphotyrosine (anti-PY) antibodies. The results show a clear decrease in tyrosine phosphorylated Etk upon Etp overexpression (Fig. 2), suggesting that Etp dephosphorylates Etk in vivo. Intriguingly, we noticed that the anti-PY antibody also reacted with the recombinant Etp (Fig. 2), indicating that some of the Etp was tyrosine phosphorylated. We also noted that the blotted Etp regained some of its acid-phosphatase activity during the blot development to catalyze autodephosphorylation (Fig. S1). To reduce the autodephosphorylation activity, blots were developed at pH 8 to inhibit Etp (Fig. S2). To exclude the possibility that the anti-PY antibody recognized Etp by nonspecific cross-reaction, we included the PY competitive inhibitor phenylphosphate that abolished the antibody interaction with Etk (Fig. S3). Taken together, our results show that Etp becomes tyrosine phosphorylated in vivo and that it catalyzes autodephosphorylation.

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