We produced WT GST Tir that was purified using GSH beads and treated with PreScission enzyme, which excised Tir and at the same table 1 time removed the GST tag. This Tir protein was used as the input in pull down experiments with GST cortactin. The first line of Fig. 3A shows that cortactin binds Tir in vitro. To map the domains involved in the interaction, we per formed pull down experiments using cortactin mutants as follows full length W525K, the N terminus, and the isolated SH3 domain. GST was used as a neg ative control. In agreement with our initial hypothesis, the isolated SH3 domain of cortactin bound Tir. However, the N terminal domain of cortactin also bound Tir. This unforeseen interaction was confirmed in experiments with cortactin carrying the point mutation, W525K in the SH3 domain.
We obtained similar results using as input the Tir phospho mimicking mutant TirY474D. Next we tested the cort actin S405,418D and Y421,466,482D mutants which were similar to the WT form in their ability Inhibitors,Modulators,Libraries to bind both Tir and TirD. These results demonstrate that cortactin and Tir interact directly in vitro, that this interaction involves both the N terminal part and the SH3 domain, and that it appears to be inde pendent of cortactin phosphorylation. Given the direct interaction between Tir and cortactin, we wondered whether Tir can activate the ability of cortactin to promote Arp23 mediated Inhibitors,Modulators,Libraries 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 both SD and 3D mutants. Similar results were 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 Inhibitors,Modulators,Libraries of the Arp23 complex. As a negative con trol we used naked beads that showed no activation. Conversely, experiments in which cortactin and its mutants were coupled to GSH beads showed similar results. These results indicate that Tir Inhibitors,Modulators,Libraries activates the ability Inhibitors,Modulators,Libraries of cortactin to promote Arp23 medi ated actin polymerization in vitro. Cortactin binding to Tir in N WASP deficient cells infected by EPEC Because cortactin binds directly both Tir and N WASP, we analyze cortactin Tir interaction in N WASP deficient cells.
Since those cells do not form pedes tals, we wondered if Tir would be present Perifosine structure at similar levels to WT cells. To address this question, we used a pre viously described fractionation protocol that enriches in Tir containing membranes. As shown in Fig. 4A, as expected Tir was enriched in the pellets compared to supernatants, as detected by west ern blotting with anti Tir mAb.