Fig. 2A shows that EPEC induces phosphorylation www.selleckchem.com/products/Vorinostat-saha.html of tyro sine 466 at 3 hours of infection in WT MEFs, as detected using an antibody against phospho Y466 cortactin. This result was corroborated using a second phospho specific antibody. Unexpectedly, phos phorylation of tyrosine residue 466 was not induced in N WASP deficient cells. This result suggests that tyrosine phosphorylation of cortactin during EPEC infection depends on the presence of N WASP. To verify this, we infected R cells with EPEC and examined levels of phosphoY466 cortactin. Fig. 2A shows Inhibitors,Modulators,Libraries that N WASP re expression partially restored cortactin tyrosine phosphor ylation levels. In three independent experiments the nor malized average induction was 10. 2 for WT cells, 0 for N WASP deficient cells and 0. 50. 1 for R cells.

This sup ports the idea that EPEC induced tyrosine Inhibitors,Modulators,Libraries phosphoryla tion of cortactin in cells requires N WASP. Given the absence of cortactin tyrosine phosphorylation in EPEC infected N Inhibitors,Modulators,Libraries WASP deficient cells, we then checked Src activation, using a commercially available phospho active Src antibody. Fig. 2B demonstrated that equal activation of Src was achieved during EPEC infec tion in all cell types studied, while, as expected, the levels of total Src remained constant during infection. This result showed that the lack of cortactin phosphorylation in N WASP deficient cells was not due to a block in Src activa tion. As a further control, Inhibitors,Modulators,Libraries 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 used a phospho specific monoclonal antibody that detects the activated form of Erk12. EPEC induced the activa tion of Erk on WT MEFs, in agreement with a previous report on T84 epithelial cells. How ever, infection of N WASP deficient cells showed reduced activation of Erk which was recovered in R cells. This result implies that Erk is activated Inhibitors,Modulators,Libraries by EPEC and may phos phorylate cortactin in vivo. More importantly, N WASP is absolutely required for the induction of Erk activation at 3 hours of infection. However, WT MEFs treated with ERK inhibitors PD98056 or U0126 showed no difference in the number of pedestals formed.

Tir binds cortactin and induces the latter to nucleate actin in vitro through an Arp23 complex mediated pathway The bacterial protein called Tir initiates what is considered to be the principal signaling cascade, which consists of Tir http://www.selleckchem.com/products/Enzastaurin.html clustering and concomitant phosphorylation on its tyro sine 474, which then recruits Nck. The latter presumably binds N WASP to initiate Arp23 complex mediated actin polymerization. We wanted to gain insights into how cortactin functions in pedestal signaling. Our initial hypothesis was that cortactin and Tir interact directly. Therefore we used the Scansite database to search for motifs in the Tir sequence to which cortactin SH3 domain could bind. We found a consensus motif centered on proline 20 of Tir.