Insulin receptor tyrosine kinase substrate links the E. coli O157:H7 actin assembly effectors Tir and EspF(U) during pedestal formation

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 05/2009; 106(16):6754-9. DOI: 10.1073/pnas.0809131106
Source: PubMed


Enterohemorrhagic Escherichia coli O157:H7 translocates 2 effectors to trigger localized actin assembly in mammalian cells, resulting in filamentous actin "pedestals." One effector, the translocated intimin receptor (Tir), is localized in the plasma membrane and clustered upon binding the bacterial outer membrane protein intimin. The second, the proline-rich effector EspF(U) (aka TccP) activates the actin nucleation-promoting factor WASP/N-WASP, and is recruited to sites of bacterial attachment by a mechanism dependent on an Asn-Pro-Tyr (NPY(458)) sequence in the Tir C-terminal cytoplasmic domain. Tir, EspF(U), and N-WASP form a complex, but neither EspF(U) nor N-WASP bind Tir directly, suggesting involvement of another protein in complex formation. Screening of the mammalian SH3 proteome for the ability to bind EspF(U) identified the SH3 domain of insulin receptor tyrosine kinase substrate (IRTKS), a factor known to regulate the cytoskeleton. Derivatives of WASP, EspF(U), and the IRTKS SH3 domain were capable of forming a ternary complex in vitro, and replacement of the C terminus of Tir with the IRTKS SH3 domain resulted in a fusion protein competent for actin assembly in vivo. A second domain of IRTKS, the IRSp53/MIM homology domain (IMD), bound to Tir in a manner dependent on the C-terminal NPY(458) sequence, thereby recruiting IRTKS to sites of bacterial attachment. Ectopic expression of either the IRTKS SH3 domain or the IMD, or genetic depletion of IRTKS, blocked pedestal formation. Thus, enterohemorrhagic E. coli translocates 2 effectors that bind to distinct domains of a common host factor to promote the formation of a complex that triggers robust actin assembly at the plasma membrane.

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    • "These proteins link to Tir via their N-terminal IMD domains, which also form 'zeppelin'-shaped homodimers associated with convex membrane protrusions (Vingadassalom et al., 2009; Weiss et al., 2009; de Groot et al., 2011; Zhao et al., 2011). The C-terminal regions of IRTKS and IRSp53 contain additional motifs, such as an SH3 domain, that link their membrane deforming activity to signalling proteins and actin regulators (Vingadassalom et al., 2009; Weiss et al., 2009). A paradox in this Tir NPY-mediated pedestal pathway is that it promotes pedestal formation with low efficiency for EPEC, yet with high efficiency for EHEC (Campellone and Leong, 2005; Campellone et al., 2006). "
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    • "(B) EspF U W33 establishes a T-shaped edge-to-face arrangement with IRTKS W378. (C) Schematic showing a single repeat of EspF U C. The N-WASP binding helix ''H'' (Cheng et al., 2008) and IRTKS binding ''P'' (Weiss et al., 2009; Vingadassalom et al., 2009) domains are indicated. The asterisk indicates the site of the W33A mutation. "
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