Effect of protein kinase-A mediated phosphorylation on the structure and association properties of the enteropathogenic Escherichia coli Tir virulence protein

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  2. Dr Mark Banfield
  3. Dr David Scott
  4. Professor Brendan Kenny
Author(s)Hawrani AS, Dempsey CE, Banfield MJ, Scott DJ, Clarke AR, Kenny B
Publication type Article
JournalJournal of Biological Chemistry
Year2003
Volume278
Issue28
Pages25839-25846
ISSN (print)0021-9258
ISSN (electronic)1083-351X
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Enteropathogenic Escherichia coli virulence is dependent on delivery of the translocated intimin receptor protein (Tir) into host cells. Tir phosphorylation on a single tyrosine (Tyr-474) is essential in mediating cytoskeletal rearrangement correlated with disease. Tir is also phosphorylated on other residues, with cAMP-dependent kinase (PKA) modification shown to play a role in Tir function. However, the mechanism by which nontyrosine phosphorylation affects Tir function remains unclear. In this study, analytical ultracentrifugation, SDS and native gel electrophoresis revealed that both Tir and its C-terminal domain (residues 385–550 of Tir that include the PKA substrate sites) exist in an equilibrium of monomers, dimers, and in the case of Tir, higher oligomers. PKA phosphorylation (1:300, PKA/C-Tir, mol/mol) shifted the equilibrium of C-Tir, but not Tir, predominantly to the dimeric state, whereas, at 100-fold higher concentrations of PKA the phosphorylated C-Tir was largely monomeric. This monomeric state was also produced at the lower PKA concentration and physiological ionic strength. Phosphorylation-mediated effects were achieved without significant changes in secondary structure as determined by circular dichroism spectroscopy. The data presented indicate that PKA-mediated phosphorylation induces changes in the association properties of the C-terminal domain of Tir that may facilitate Tir-Tir interactions and subsequently C-Tir-host protein interactions in vivo.
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc.
URLhttp://dx.doi.org/10.1074/jbc.M212409200
DOI10.1074/jbc.M212409200
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