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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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Identification of the phosphorylated Etp residue.(A) The region containing the DPY motif (in bold) of Etp is compared with the corresponding region of several members of the LMW-PTP family. They include AmsI of Erwinia amylovora, the E. coli-encoded Etp paralog Wzb, Yco5 of Klebsiella pneumoniae, EspP of Ralstonia solanacearum, the human PA1F protein, and the bovine PPAC protein encoded by the ACP1 gene. Numbering is according to the Etp sequence. (B) Homology model of the Etp structure in the absence of bound substrate based on the NMR structure of Wzb [19]. Side chains Tyr121 and Asp119 are shown in sticks. The phosphate binding loop (yellow) and catalytic cysteine C13 are in the center. (C) EPEC mutant Δetp::kan was transformed with plasmids expressing different Etp mutants all of which had C-terminal 6His tags. Proteins were extracted from the different cultures and were subjected to Western blot analysis with anti-Etk, anti-6His and anti-PY antibodies as indicated. The corresponding strain is indicated above each of the lanes. (D) In vitro kinetics of Etp and variants with phosphorylated MBP-Etk as substrate. The graph plots the rate of inorganic phosphate produced versus substrate concentration for Etp, EtpY121F, and EtpD119E. Kinetic constants are in Table 1.
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pone-0037984-g003: Identification of the phosphorylated Etp residue.(A) The region containing the DPY motif (in bold) of Etp is compared with the corresponding region of several members of the LMW-PTP family. They include AmsI of Erwinia amylovora, the E. coli-encoded Etp paralog Wzb, Yco5 of Klebsiella pneumoniae, EspP of Ralstonia solanacearum, the human PA1F protein, and the bovine PPAC protein encoded by the ACP1 gene. Numbering is according to the Etp sequence. (B) Homology model of the Etp structure in the absence of bound substrate based on the NMR structure of Wzb [19]. Side chains Tyr121 and Asp119 are shown in sticks. The phosphate binding loop (yellow) and catalytic cysteine C13 are in the center. (C) EPEC mutant Δetp::kan was transformed with plasmids expressing different Etp mutants all of which had C-terminal 6His tags. Proteins were extracted from the different cultures and were subjected to Western blot analysis with anti-Etk, anti-6His and anti-PY antibodies as indicated. The corresponding strain is indicated above each of the lanes. (D) In vitro kinetics of Etp and variants with phosphorylated MBP-Etk as substrate. The graph plots the rate of inorganic phosphate produced versus substrate concentration for Etp, EtpY121F, and EtpD119E. Kinetic constants are in Table 1.

Mentions: Comparison of Etp to other LMW-PTPs associated with polysaccharide production highlighted only one conserved tyrosine residue, Tyr121, located in the conserved DPY motif (Fig. 3A, B) that also included the essential catalytic acid Asp119. To test whether Tyr121 is the Etp phosphorylation site, we constructed plasmids expressing Etp mutants. Prior to plasmid construction, we determined the actual N-terminus of Etp, since early versions of the EPEC genome sequence annotated the etp ORF starting at a GTG codon, 5 codons prior to an ATG codon. To resolve which codon was the native translation start site, we cloned etp including the 80 bp upstream of the putative GTG initiation codon that contained the etp native ribosomal binding site. In addition, we added a hexahistidine (6His) tag to the 3′ end of the etp ORF followed by a stop codon. The generated plasmid pCNY506, encoding Etp-6His, was introduced into E. coli and the expressed Etp-6His was purified by affinity chromatography. The native N-terminal sequence of the purified protein was determined to be MAQLKFNSILVVZ. These results indicated that Etp translation starts at the first ATG of the ORF rather than the GTG. We thus termed the ATG encoded residue as Met1. We further used this vector to generate Etp mutants in the DPY motif.


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)

Identification of the phosphorylated Etp residue.(A) The region containing the DPY motif (in bold) of Etp is compared with the corresponding region of several members of the LMW-PTP family. They include AmsI of Erwinia amylovora, the E. coli-encoded Etp paralog Wzb, Yco5 of Klebsiella pneumoniae, EspP of Ralstonia solanacearum, the human PA1F protein, and the bovine PPAC protein encoded by the ACP1 gene. Numbering is according to the Etp sequence. (B) Homology model of the Etp structure in the absence of bound substrate based on the NMR structure of Wzb [19]. Side chains Tyr121 and Asp119 are shown in sticks. The phosphate binding loop (yellow) and catalytic cysteine C13 are in the center. (C) EPEC mutant Δetp::kan was transformed with plasmids expressing different Etp mutants all of which had C-terminal 6His tags. Proteins were extracted from the different cultures and were subjected to Western blot analysis with anti-Etk, anti-6His and anti-PY antibodies as indicated. The corresponding strain is indicated above each of the lanes. (D) In vitro kinetics of Etp and variants with phosphorylated MBP-Etk as substrate. The graph plots the rate of inorganic phosphate produced versus substrate concentration for Etp, EtpY121F, and EtpD119E. Kinetic constants are in Table 1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366997&req=5

pone-0037984-g003: Identification of the phosphorylated Etp residue.(A) The region containing the DPY motif (in bold) of Etp is compared with the corresponding region of several members of the LMW-PTP family. They include AmsI of Erwinia amylovora, the E. coli-encoded Etp paralog Wzb, Yco5 of Klebsiella pneumoniae, EspP of Ralstonia solanacearum, the human PA1F protein, and the bovine PPAC protein encoded by the ACP1 gene. Numbering is according to the Etp sequence. (B) Homology model of the Etp structure in the absence of bound substrate based on the NMR structure of Wzb [19]. Side chains Tyr121 and Asp119 are shown in sticks. The phosphate binding loop (yellow) and catalytic cysteine C13 are in the center. (C) EPEC mutant Δetp::kan was transformed with plasmids expressing different Etp mutants all of which had C-terminal 6His tags. Proteins were extracted from the different cultures and were subjected to Western blot analysis with anti-Etk, anti-6His and anti-PY antibodies as indicated. The corresponding strain is indicated above each of the lanes. (D) In vitro kinetics of Etp and variants with phosphorylated MBP-Etk as substrate. The graph plots the rate of inorganic phosphate produced versus substrate concentration for Etp, EtpY121F, and EtpD119E. Kinetic constants are in Table 1.
Mentions: Comparison of Etp to other LMW-PTPs associated with polysaccharide production highlighted only one conserved tyrosine residue, Tyr121, located in the conserved DPY motif (Fig. 3A, B) that also included the essential catalytic acid Asp119. To test whether Tyr121 is the Etp phosphorylation site, we constructed plasmids expressing Etp mutants. Prior to plasmid construction, we determined the actual N-terminus of Etp, since early versions of the EPEC genome sequence annotated the etp ORF starting at a GTG codon, 5 codons prior to an ATG codon. To resolve which codon was the native translation start site, we cloned etp including the 80 bp upstream of the putative GTG initiation codon that contained the etp native ribosomal binding site. In addition, we added a hexahistidine (6His) tag to the 3′ end of the etp ORF followed by a stop codon. The generated plasmid pCNY506, encoding Etp-6His, was introduced into E. coli and the expressed Etp-6His was purified by affinity chromatography. The native N-terminal sequence of the purified protein was determined to be MAQLKFNSILVVZ. These results indicated that Etp translation starts at the first ATG of the ORF rather than the GTG. We thus termed the ATG encoded residue as Met1. We further used this vector to generate Etp mutants in the DPY motif.

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