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Phosphorylation of the enteropathogenic E. coli receptor by the Src-family kinase c-Fyn triggers actin pedestal formation

Nature Cell Biology volume 6, pages 618–625 (2004)Cite this article

Abstract

Enteropathogenic Escherichia coli (EPEC) causes diarrhoeal disease worldwide1. Pathogen adherence to host cells induces reorganization of the actin cytoskeleton into 'pedestal-like' pseudopods beneath the extracellular bacteria2. This requires two bacterial virulence factors that mimic a ligand-receptor interaction. EPEC delivers its own receptor, the translocated intimin receptor (Tir), into the target cell plasma membrane, which is phosphorylated on interaction with the bacterial surface protein intimin3. Tir phosphorylated on Tyr 474 (ref. 4) binds the cellular adaptor Nck5,6, triggering actin polymerization7. Nevertheless, despite its critical role, the mechanism of Tir Tyr 474 phosphorylation remains unknown. Here, by artificially uncoupling Tir delivery and activity, we show that Tir phosphorylation and Nck-dependent pedestal formation require the Src-family kinase (SFK) c-Fyn. SFK inhibitors prevent Tyr 474 phosphorylation, and cells lacking c-fyn are resistant to pedestal formation. c-Fyn exclusively phosphorylates clustered Tir in vitro, and kinase knockdown suppresses Tir phosphorylation and pedestal formation in cultured cells. These results identify the transient interaction with host c-Fyn as a pivotal link between bacterial Tir and the cellular Nck–WASP–Arp2/3 cascade, illuminating a tractable experimental system in which to dissect tyrosine kinase signalling.

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Figure 1: Clustering is required for Tir Tyr 474 phosphorylation.
Figure 2: Src family kinase inhibitors prevent Tir phosphorylation and actin pedestal formation in cultured cells.
Figure 3: Tir is a c-Fyn substrate both in cultured cells and in vitro.
Figure 4: Silencing of c-Fyn expression attenuates pedestal formation and Tir phosphorylation in cultured cells.

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Acknowledgements

We thank J. Leong, D. Tipper, E. Koronakis and C. Hughes for discussions, C. ffrench-Constant and H. Colognato for discussions and advice on siRNA, and M. Way for the gift of kinase-knockout cell lines and helpful advice. This work was supported by a Wellcome Trust programme grant to V.K. and a Biotechnology and Biological Sciences Research Council studentship to N.P.

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  1. Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK

    Neil Phillips, Richard D. Hayward & Vassilis Koronakis

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Correspondence to Vassilis Koronakis.

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Phillips, N., Hayward, R. & Koronakis, V. Phosphorylation of the enteropathogenic E. coli receptor by the Src-family kinase c-Fyn triggers actin pedestal formation. Nat Cell Biol 6, 618–625 (2004). https://doi.org/10.1038/ncb1148

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