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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

A model for the role of Etp and Etk in capsule formation.Cycling of Etk phosphorylation and dephosphorylation are required for capsule production (arrow 1). This cycling is mediated by the autokinase and autophosphatase activities of Etk and Etp, respectively (arrows 2 and 3). Etp also cycles between phosphorylated and unphosphorylated forms and this cycling is catalyzed by Etp autodephosphorylation (arrow 4) and a yet to be defined kinase (arrow 5). Etp cycling per se is not required for capsule formation. However, based on our results we hypothesize that the unphosphorylated Etp has an inhibitory effect on capsule formation (arrow 6), which can be removed by Etp phosphorylation.
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pone-0037984-g007: A model for the role of Etp and Etk in capsule formation.Cycling of Etk phosphorylation and dephosphorylation are required for capsule production (arrow 1). This cycling is mediated by the autokinase and autophosphatase activities of Etk and Etp, respectively (arrows 2 and 3). Etp also cycles between phosphorylated and unphosphorylated forms and this cycling is catalyzed by Etp autodephosphorylation (arrow 4) and a yet to be defined kinase (arrow 5). Etp cycling per se is not required for capsule formation. However, based on our results we hypothesize that the unphosphorylated Etp has an inhibitory effect on capsule formation (arrow 6), which can be removed by Etp phosphorylation.

Mentions: Based on our results we propose the model shown in Fig. 7. This model and our study raise several new questions. First, why does EtpY121F with near wild-type catalytic activity inhibit capsule formation? One possibility is that the Y121F substitution alters the enzyme's substrate specificity. If this is the case, the affected substrate is likely not Etk, since our results showed that EtpY121F dephosphorylated Etk as efficiently as wild-type Etp. UDP-glucose dehydrogenase (Ugd) has been described to be a substrate of Wzc/Wzb proteins in E. coli[14]. By analogy, if Etp dephosphorylates Ugd, the Y121F substitution could affect substrate recognition of Ugd. Why Etp phosphorylation alleviates this inhibitory effect must also be addressed. A second possibility is that Phe121 affects the interaction of EtpY121F with other Etp molecules or with another protein affecting its function. Interestingly, a Bacillus subtilis LMW-PTP, whose sequence contains Phe in place of Etp's Tyr121, was observed by NMR to oligomerize at sub-millimolar concentrations [21].


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)

A model for the role of Etp and Etk in capsule formation.Cycling of Etk phosphorylation and dephosphorylation are required for capsule production (arrow 1). This cycling is mediated by the autokinase and autophosphatase activities of Etk and Etp, respectively (arrows 2 and 3). Etp also cycles between phosphorylated and unphosphorylated forms and this cycling is catalyzed by Etp autodephosphorylation (arrow 4) and a yet to be defined kinase (arrow 5). Etp cycling per se is not required for capsule formation. However, based on our results we hypothesize that the unphosphorylated Etp has an inhibitory effect on capsule formation (arrow 6), which can be removed by Etp phosphorylation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0037984-g007: A model for the role of Etp and Etk in capsule formation.Cycling of Etk phosphorylation and dephosphorylation are required for capsule production (arrow 1). This cycling is mediated by the autokinase and autophosphatase activities of Etk and Etp, respectively (arrows 2 and 3). Etp also cycles between phosphorylated and unphosphorylated forms and this cycling is catalyzed by Etp autodephosphorylation (arrow 4) and a yet to be defined kinase (arrow 5). Etp cycling per se is not required for capsule formation. However, based on our results we hypothesize that the unphosphorylated Etp has an inhibitory effect on capsule formation (arrow 6), which can be removed by Etp phosphorylation.
Mentions: Based on our results we propose the model shown in Fig. 7. This model and our study raise several new questions. First, why does EtpY121F with near wild-type catalytic activity inhibit capsule formation? One possibility is that the Y121F substitution alters the enzyme's substrate specificity. If this is the case, the affected substrate is likely not Etk, since our results showed that EtpY121F dephosphorylated Etk as efficiently as wild-type Etp. UDP-glucose dehydrogenase (Ugd) has been described to be a substrate of Wzc/Wzb proteins in E. coli[14]. By analogy, if Etp dephosphorylates Ugd, the Y121F substitution could affect substrate recognition of Ugd. Why Etp phosphorylation alleviates this inhibitory effect must also be addressed. A second possibility is that Phe121 affects the interaction of EtpY121F with other Etp molecules or with another protein affecting its function. Interestingly, a Bacillus subtilis LMW-PTP, whose sequence contains Phe in place of Etp's Tyr121, was observed by NMR to oligomerize at sub-millimolar concentrations [21].

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