Provided the direct interaction involving Tir and cortactin, we wondered no matter whether Tir can activate the capability of cortactin to promote Arp2 3 mediated actin polymerization. We coupled recombinant Tir protein to 1M beads, and then we washed the beads with Xb buffer and blocked them in Xb buffer containing 1% BSA. Next we incubated them with purified Arp and actin in Xb buffer containing WT and cortactin mutants. Fig. 3C shows that Tir activated WT cortactin and each SD and 3D mutants. Similar results had been obtained for TirD. The W525K mutant was also activated, although weakly. As expected, W22A cortactin was not activated, indicating that the effect was mediated by cort actin activation of the Arp2 3 complex. As a damaging con trol we used naked beads that showed no activation.
selleckchem Conversely, experiments in which cortactin and its mutants have been coupled to GSH beads showed equivalent outcomes. These results indicate that Tir activates the capability of cortactin to promote Arp2 3 medi ated actin polymerization in vitro. Cortactin binding to Tir in N WASP deficient cells infected by EPEC Since cortactin binds straight both Tir and N WASP, we analyze cortactin Tir interaction in N WASP deficient cells. Because these cells usually do not type pedes tals, we wondered if Tir will be present at related levels to WT cells. To address this question, we applied a pre viously described fractionation protocol that enriches in Tir containing membranes. As shown in Fig. 4A, as expected Tir was enriched inside the pellets compared to supernatants, as detected by west ern blotting with anti Tir mAb.
We observed that a band with slower electrophoretic motility was the predominant type of Tir in the pellets, which represents fully modified Tir. WT and N WASP deficient cells presented detect able amounts of mature Tir that was slightly decreased on R cells. FL cortactin features a closed conformation. Thus, selleck chemical we decided to make use of N terminal cortactin, the SH3 domain and GST as a damaging handle to execute pull down experiments with lysates of EPEC infected and uninfected WT, N WASP deficient and R cells. Western blotting in Fig. 4B shows that NH2 bound Tir in EPEC infected but not uninfected cells, with no appreciable dif ferences involving WT, N WASP and R cells. Comparable outcomes have been obtained with total cell lysates despite the fact that longer expo confident occasions where essential to detect Tir.
In contrast, neither the isolated SH3 domain nor the GST adverse manage bound Tir in any on the cells types used. In view of those final results, we can conclude that in cells, cort actin binds Tir mainly via its N terminal area. To test whether or not the SH3 domain of cortactin prefers to bind N WASP over Tir, we performed pull downs with clarified total lysates, and we then stripped and reprobed the blots with anti N WASP antibody.