Effect of protein kinase A-mediated phosphorylation on the structure and association properties of the enteropathogenic Escherichia coli Tir virulence protein.
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.
Departments of Pathology and Microbiology and Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
Ayman
A
Hawrani
Christopher E
CE
Dempsey
Mark J
MJ
Banfield
David J
DJ
Scott
Anthony R
AR
Clarke
Brendan
B
Kenny
D002942:Circular Dichroism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000378:metabolism*; D019791:Guanidine / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000378:metabolism; D014461:Ultracentrifugation
D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; D014443:Tyrosine; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases; D019791:Guanidine