As a further handle, we treated the cells with per vanadate and observed robust phosphorylation of cortactin tyrosine 466. Similarly we sought to establish the activation status of Erk in EPEC infected cells. We utilised a phospho distinct monoclonal antibody that detects the activated type of Erk1 two. EPEC induced the activa tion of Erk on WT MEFs, in agreement having a prior report on T84 epithelial cells. How ever, infection of N WASP deficient cells showed decreased activation of Erk which was recovered in R cells. This result implies that Erk is activated by EPEC and may possibly phos phorylate cortactin in vivo. Much more importantly, N WASP is totally essential for the induction of Erk activation at three hours of infection. Even so, WT MEFs treated with ERK inhibitors PD98056 or U0126 showed no difference within the number of pedestals formed.
Tir binds cortactin and induces the latter to nucleate actin in vitro by way of Midostaurin an Arp2 three complicated mediated pathway The bacterial protein named Tir initiates what exactly is regarded to become the principal signaling cascade, which consists of Tir clustering and concomitant phosphorylation on its tyro sine 474, which then recruits Nck. The latter presumably binds N WASP to initiate Arp2 3 complicated mediated actin polymerization. We wanted to achieve insights into how cortactin functions in pedestal signaling. Our initial hypothesis was that cortactin and Tir interact directly. Consequently we applied the Scansite database to search for motifs within the Tir sequence to which cortactin SH3 domain could bind. We identified a consensus motif centered on proline 20 of Tir.
We initial performed pull down experiments with purified recombinant Tir and cortactin proteins. We made WT GST Tir that was purified making use of GSH beads and treated with Odanacatib structure PreScission enzyme, which excised Tir and at the same time removed the GST tag. This Tir protein was made use of because the input in pull down experiments with GST cortactin. The initial line of Fig. 3A shows that cortactin binds Tir in vitro. To map the domains involved within the interaction, we per formed pull down experiments applying cortactin mutants as follows, complete length W525K, the N terminus, and also the isolated SH3 domain. GST was applied as a neg ative control. In agreement with our initial hypothesis, the isolated SH3 domain of cortactin bound Tir. Nevertheless, 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 comparable final results working with as input the Tir phospho mimicking mutant TirY474D. Next we tested the cort actin S405,418D and Y421,466,482D mutants which were comparable towards the WT type in their ability to bind both Tir and TirD. These final results demonstrate that cortactin and Tir interact directly in vitro, that this interaction requires both the N terminal part along with the SH3 domain, and that it seems to become inde pendent of cortactin phosphorylation.