Further experiments are needed to address this question. Finally, in our effort to understand what kind of com plexes form in vivo, we considered all available in vitro data concerning the interaction of Tir, under Nck, N WASP and cort actin. Inhibitors,Modulators,Libraries Thus Nck binds cortactin only when phosphor ylated by Src, through an interaction between the phosphotyrosine and the SH2 domain. Therefore since Tir and Nck interact through the single SH2 domain of Nck, formation of a Tir Nck cortactin complex appears to be impossible. Cortactin phosphorylated by Src is not able to interact with N WASP, as shown with recombinant proteins and further corroborated in the two hybrid assay. That adds to the evidence against the possibil ity that cortactin bridges both proteins, i. e. Nck cortactin N WASP.

This leaves three possible types of complexes Tir Nck N WASP cortactin, Tir cortactin N WASP and Tir cortactin. Given the fact that reducing of cortactin expression with siRNA inhibits pedestal formation, that EPEC infection induces cortactin phosphorylation in an N WASP dependent fash ion, and that Tir binds and activates cortactin Inhibitors,Modulators,Libraries we conclude that cortactin contributes to the Tir Nck N WASP pathway, possibly by regulating N WASP activ ity. In other words, cortactin and N WASP would act in a complex in this scenario. If we envision Inhibitors,Modulators,Libraries pedestals as a dynamic actin structure, and in fact pedestal motility has been shown, then it is reasonable to think that pro teins promoting actin polymerization would act in a cyclic manner. We speculate that cortactin is a cycling switch for N WASP in pedestals.

Inhibitors,Modulators,Libraries Deletion of Tir abrogates pedestal formation by EPEC implying that Tir mediates the major but not only path way for actin assembly in pedestals. Indeed, elegant work has shown that the EPEC effector protein EspF directly activates N WASP. We can not exclude that cortactin participates in this pathway. Conclusion The function of cortactin in pedestals, and how its func tion is regulated, seems to differ between EPEC and EHEC. EHEC induces tyrosine dephosphorylation of cortactin whereas EPEC induces its tyrosine phophorylation. During EHEC infection, the Tir cort actin interaction was mapped to the N terminal region of both molecules, but only cortactin phosphorylated by Src bound to TirEHEC.

In our study, cortactin bound directly to TirEPEC independ ently of phosphorylation since cortactin mutants mimick ing phosphorylation by Erk and Src interacted with Tir, and were activated to a Inhibitors,Modulators,Libraries similar extent in vitro. This finding further supports our results using EPEC infected cells that show that the interaction between Tir and cortactin is mediated through the N terminal part of the cortactin selleck chem Paclitaxel molecule. Our results are compatible with the formation of complexes in which cortactin may interact with Tir via its N terminal domain and with N WASP via its SH3 domain.