title: Clustering transfers the translocated Escherichia coli receptor into lipid rafts to stimulate reversible activation of c-Fyn.
abstract: Enteropathogenic Escherichia coli (EPEC) mimic a ligand-receptor interaction to induce 'pedestal-like' pseudopodia on mammalian cells, providing a tractable system to study tyrosine kinase signalling to the actin cytoskeleton. EPEC delivers its own receptor (Tir), which is engaged by a bacterial surface ligand (intimin). When Tir delivery and activity are uncoupled, intimin-induced Tir clustering stimulates Tir(Y474) phosphorylation by the Src-family kinase (SFK) c-Fyn, triggering actin polymerization and pedestal formation. How c-Fyn specifically targets Tir and is regulated remains unknown. We show that clustering transfers Tir into cholesterol-rich detergent-resistant microdomains (DRMs), a signal prompting transient c-Fyn accumulation at bacterial adhesion sites. Co-clustering of Tir(Y474) and c-Fyn in DRMs rapidly stimulates robust kinase activation both by induced c-Fyn(Y531) dephosphorylation to unlock the inactive state and by reciprocal c-Fyn(Y417) autophosphorylation to promote activity. After signal induction, c-Fyn dissipates and the resting state restored by Csk-dependent phosphorylation of c-Fyn(Y531). These data illustrate a sophisticated mechanism evolved by a pathogen effector to reversibly regulate SFKs, and resolve early interactions at a model receptor initiating tyrosine kinase signalling.
authors.affiliation: University of Cambridge, Department of Pathology, Cambridge, UK.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Hayward
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hume
authors.forename: Daniel
authors.initials: D
authors.lastname: Humphreys
authors.forename: Neil
authors.initials: N
authors.lastname: Phillips
authors.forename: Katherine
authors.initials: K
authors.lastname: Smith
authors.forename: Vassilis
authors.initials: V
authors.lastname: Koronakis
mesh_terms: D000818:Animals; D001422:Bacterial Adhesion; D002460:Cell Line; D054308:Enteropathogenic Escherichia coli / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000382:microbiology; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D051076:Proto-Oncogene Proteins c-fyn / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015398:Signal Transduction; D015854:Up-Regulation
chemical_list: D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; D051076:Proto-Oncogene Proteins c-fyn