This binding induces the clustering of Tir, followed by its phosphorylation on tyro sine residue 474 in the cytoplasmic C terminal domain. The phosphotyrosine selleck chemical moiety recruits the host cell adaptor protein Nck, which binds and presumably activates N WASP, leading Inhibitors,Modulators,Libraries to actin polymerization mediated by the Arp23 complex. Although this pathway is recognized as the principal one operating in EPEC, another Nck inde pendent pathway has also been described in these bacteria. Furthermore, the complexity of EPEC signal transduc tion is not fully understood. Tir is inserted in the cell membrane, where it adopts a hairpin loop structure, with both N and C termini project ing into the host cytoplasm. Pedestals are dynamic structures that undergo constant remodeling by cycles of actin polymerizationdepolymerization.

It is impor tant to understand the contribution of other signals to pedestal formation, not only for EPEC but also for other actin based processes. For instance, it has been postulated that Tir Nck signaling mimics the nephrin Nck actin pathway. Cortactin is a key regulator of the actin cytoskeleton which Inhibitors,Modulators,Libraries plays a crucial role in cell invasion and actin based motility during the infection of many microbial patho gens. Cortactin possesses an N terminal acidic domain which harbors a DDW motif that activates, albeit weakly, the Arp23 complex at branching points. The NTA domain is followed by a series of repeat domains that bind filamentous actin. The C ter minal SH3 domain of cortactin binds various pro teins, such as N WASP, which is a ubiquitously expressed member of the WASP family of proteins.

Cortactin can be phosphor ylated by tyrosine kinases and serinethreonine kinases. Src kinase targets tyrosine residues 421, 466 and 482 while Erk phos phorylates serines 405 and 418 which lie in a proline Inhibitors,Modulators,Libraries rich area. Interestingly, a Src family member and Abl kinases phosphorylate Tir. The Arp23 complex can be independently activated to initiate actin polymerization by the VCA domain of WASP members and by both the NTA and F actin binding repeats of cortactin. Theoreti cally N WASP, cortactin and the Arp23 complex can form ternary complexes. Cortactin has been shown in vitro to bind and activate N WASP via an SH3 proline rich domain interaction. This activation is regulated pos itively and negatively when cortactin Inhibitors,Modulators,Libraries is phosphorylated by Erk and Src respectively.

Erk phosphorylation of cortactin or the double mutation S405,418D in cortactin that mim ics this phosphorylation enhance the proteins binding to and activation of N WASP. Conversely, Src phosphoryla tion inhibits the ability of both Erk phosphorylated Inhibitors,Modulators,Libraries cort actin, and that doubly mutated S405,418D cortactin, to activate N WASP. Furthermore, phospho mimetic muta sellectchem tion of the three tyrosine residues targeted by Src inhibited the ability of S405,418D cort actin to activate N WASP.