Formulation of models for how EPEC initiates pedestal formation in mammalian cells has been hampered by difficulties in identifying the entire repertoire of bacterial effectors necessary for actin assembly. Expression of Tir in mammalian cells now demonstrates that no other translocated proteins are required for actin pedestal formation. Cells expressing Tir were fully capable of forming pedestals after challenge with an E. coli K-12 strain that expresses intimin, but that does not translocate any bacterial molecules into the host cell. Actin pedestals formed on transfected cells were morphologically similar to those formed on cells after translocation of the entire repertoire of EPEC effectors, contained the same principle host factors, and were generated at an indistinguishable rate and efficiency. Therefore, translocated EPEC molecules other than Tir play at most an auxiliary role in stimulating actin pedestal formation.
A previous paper indicated that Tir expressed in mammalian cells was not modified to its mature tyrosine-phosphorylated form, suggesting that other codelivered effectors may be required for Tir to reach its completely modified state (Kenny and Warawa, 2001
). In this work, we also found that mammalian expression of Tir did not result in an observable increase in phosphotyrosine staining. However, phosphotyrosine residues colocalized with Tir after clustering, implying that oligomerization induces phosphorylation of Tir by mammalian tyrosine kinases independently of other EPEC effectors. Indeed, immunoprecipitation of Tir and Western blotting with antiphosphotyrosine antibodies confirm that clustering triggers a dramatic increase in Tir tyrosine phosphorylation (unpublished data). Although it remains possible that additional EPEC molecules facilitate serine/threonine phosphorylation of Tir, these modifications are apparently not required for Tir to stimulate localized actin assembly.
These results suggest that the central role for intimin, the only bacterial molecule other than Tir that participates directly in actin pedestal formation, is to cluster Tir in the plasma membrane, thereby triggering phosphorylation of Y474. This finding is consistent with the previously observed correlation between the ability of intimin to bind to Tir and its ability to initiate actin assembly in EPEC-primed cells (Liu et al., 2002
). Here, we demonstrate that intimin can be replaced by entirely unrelated molecules, i.e., antibodies that artificially cluster Tir. The observation that anti-HA antibodies trigger actin assembly in cells that express HA-tagged Tir indicates that Tir signaling does not even require engagement of its intimin-binding domain.
Another domain of Tir, the NH2
-terminal cytoplasmic region, binds the focal adhesion proteins α-actinin, talin, and vinculin in vitro (Freeman et al., 2000
; Goosney et al., 2000
; Huang et al., 2002
). This work reveals that these interactions do not play any observable role in pedestal formation because TirMC, which completely lacks this domain, is fully capable of signaling host cells to generate pedestals. The COOH-terminal cytoplasmic domain of Tir has also been reported to bind to α-actinin (Freeman et al., 2000
). However, this interaction is not sufficient to promote recruitment within cells because α-actinin is not detectable beneath sites of clustered TirMC(Y474F), a Tir derivative defective at actin polymerization. Because α-actinin does localize to TirMC-derived pedestals, its recruitment may be a simple consequence of localized actin assembly initiated by the COOH terminus of Tir.
The best-characterized activity of the COOH-terminal domain of Tir is its ability to bind the SH2 domain of Nck, a host adaptor protein required for EPEC pedestal formation (Gruenheid et al., 2001
; Campellone et al., 2002
). Nck is also critical for actin pedestal formation initiated by ectopically expressed TirMC, and clustering of this Tir derivative triggers recruitment of Nck, N-WASP, and the Arp2/3 actin-nucleating complex, the mammalian components necessary for pedestal formation. Moreover, we found that the minimal Nck-binding phosphopeptide derived from Tir, by itself, is sufficient to stimulate actin assembly in Xenopus
extracts after immobilization on beads. We cannot rule out the possibility that domains in the COOH terminus of Tir other than the Nck-binding sequence may also contribute to actin assembly, or that the requirements for actin polymerization in vitro may be different than at the membrane of intact cells. Nevertheless, these results support a remarkably simple model for pedestal formation, i.e., the ultimate role of the type III secretion apparatus, intimin, and Tir in actin assembly is to cluster a 12-residue Nck-binding site beneath the plasma membrane. This sequence, after being tyrosine phosphorylated by host kinases, is sufficient to trigger a signaling cascade leading to localized actin polymerization. This model implies that all other components required for actin assembly are recruited subsequent to engagement of Nck by the Tir phosphopeptide.
It is not yet clear if actin assembly is simply a consequence of a high local concentration of Nck, or whether Tir-binding by Nck somehow “activates” the adaptor for downstream signaling. Interestingly, a derivative of TirMC in which the cytoplasmic COOH terminus was replaced with Nck failed to stimulate actin assembly after clustering beneath the plasma membrane (unpublished data), and GST-Nck–coated latex beads did not trigger actin tail formation in Xenopus extracts (Ho, H., personal communication; unpublished data). These results suggest that the recruitment of Nck, specifically by Tir, may be critical for triggering localized actin assembly.
The SH2 domain of Nck also binds the tyrosine-phosphorylated membrane protein A36R of vaccinia virus, an interaction required for efficient actin tail formation by that pathogen (Frischknecht et al., 1999
). Our results suggest that recruitment of Nck by A36R, which contains a Nck-binding site nearly identical to that of Tir, may also be sufficient for initiation of actin assembly. After recruitment to the plasma membrane, the SH3 domains of Nck may interact directly with the proline-rich region of N-WASP (Rohatgi et al., 2001
), a protein required for actin assembly by both EPEC and vaccinia (Lommel et al., 2001
; Snapper et al., 2001
). However, this domain of N-WASP is dispensable for the generation of pedestals; rather, the WH1 (WASP–homology-1) domain seems to be critical for recruitment to sites of EPEC adherence (Lommel et al., 2001
). This observation suggests that EPEC signaling closely resembles the vaccinia pathway in which the interaction of Nck with N-WASP is apparently mediated by a third protein, WIP (WASP-interacting protein), which binds both to Nck and to the WH1 domain of N-WASP (Moreau et al., 2000
In addition to binding Tir and A36R, the SH2 domain of Nck binds at least 12 mammalian phosphoproteins, including many receptor tyrosine kinases such as EGFr, PDGFr, and VEGFr, as well as proteins downstream of tyrosine kinases, like Dok and IRS-3 (Buday et al., 2002
). The manner in which the signaling cascade exploited by the Tir phosphopeptide resembles pathways stimulated by engagement of these receptors remains to be determined. However, the establishment of easily manipulated experimental systems, such as Tir-expressing cells and peptide-coated beads in Xenopus
extracts, will greatly facilitate the further dissection of these pathways, leading to a more complete understanding of Nck signaling.