WT and N WASP deficient cells presented detect able amounts of mature Tir that was slightly reduced on R cells. FL cortactin www.selleckchem.com/products/chir-99021-ct99021-hcl.html has a closed conformation. Therefore, we decided to use N terminal cortactin, the SH3 domain and GST as a negative control to perform pull down experiments Inhibitors,Modulators,Libraries with lysates of EPEC infected and uninfected WT, N WASP deficient and R cells. Western blotting in Fig. 4B shows that NH2 bound Tir Inhibitors,Modulators,Libraries in EPEC infected but not uninfected cells, with no appreciable dif ferences between WT, N WASP and R cells. Similar results were obtained with total cell lysates although longer expo sure times where necessary to detect Tir. In contrast, neither the isolated SH3 domain nor the GST negative control bound Tir in any of the cells types used.

In view of these results, we can conclude that in cells, cort actin binds Tir primarily through its N terminal region. To test whether 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 Inhibitors,Modulators,Libraries the blots with anti N WASP antibody. This approach was necessary because the pellets did not contain easily detectable levels of N WASP. As previously described, the SH3 domain of cortactin was able to pull down N WASP in WT cells but not N WASP deficient cells. This argues in favor of the conclusion that the N terminal region of cortactin is involved in binding Tir, while the SH3 domain is involved in binding N WASP. Discussion Cortactin is a scaffold protein implicated in many cellular Inhibitors,Modulators,Libraries processes since it directly contributes to cytoskeleton remodeling.

Cortactin also has oncogenic properties due to its role in controlling invadopodia formation and Inhibitors,Modulators,Libraries cell migration. Moreover, cortactin has emerged as an impor tant target of numerous pathogens, including enteropath ogenic E. coli that manipulate the actin cytoskeleton in order to invade the host and propagate there. EPEC cause severe diarrheal disease in humans by colonizing the gut mucosa and producing AE lesions. EPEC attach to mammalian intestinal cells and induce reorganization of the actin cytoskeleton into pedestal like structures under neath the bacteria. A crucial event for pedestal formation is the insertion into the host cell membranes of the EPEC effector Tir, which is initially injected into the cell by a type III secretion system. Tir mimics signaling pathways of the infected cell.

Thus it can serve as a powerful model sys tem to study eukaryotic transmembrane signaling. In fact, the Tir Nck N WASP pathway is the principal one through which actin polymerizes in EPEC pedestals. Those reasons prompted us to study cortactin signaling during EPEC infection using N WASP deficient cells. Although kinase inhibitor AZD9291 cortactin localizes to pedestals and its truncated forms exert a dominant negative effect, its function is not clear.