These results higher light the fine tuned nature of cortactin regulation during EPEC and EHEC infections. Cortactin can activate the Arp2 three complicated straight by way of its NTA domain, and indirectly by utilizing its SH3 domain to activate N WASP. We wondered no matter whether the binding of Tir to cortactin would activate the latter and market Arp2 3 complicated dependent actin polymerization. As shown in Fig. 3B, Tir coated beads activated cortactin. In addition, as for the binding, the activation of cortactin by Tir was not affected by the phos phorylation status of cortactin, which further supports the concept that in EPEC signaling, Tir binds and activates cortac tin independently of the latters phosphorylation status.
At this point, we favored the conclusion that the relevant contribution underlying cortactin Tir binding occurs by means of the N terminal moiety of cortactin, because our pre vious research indicated that selleck inhibitor phosphorylation of cortactin affects primarily its interaction with partners by way of the SH3 domain. To test this hypothesis, we applied cell lysates that represent a additional restrictive situation with higher similarity to binding situations in vivo. Consistent with our reasoning, the N terminal area of cortactin bound Tir, whereas the isolated SH3 domain did not in any from the cells kind tested. In view of these final results, we are able to conclude that in cells cortactin binds Tir primarily through its N terminal region, even though the contribution in the SH3 domain seems to be irrelevant. In addition, the interaction between Tir and cortactin is independent of phosphorylation and doesn’t need N WASP, because we detected related levels of interaction in WT, N WASP defi cient and R cells.
Alternatively, the cortactin SH3 consensus web page on Tir may perhaps be occupied by other SH3 domains which include tyrosine kinases or the cortactin SH3 domain you can find out more may possess a pref erence for binding N WASP. As previously described, the SH3 domain of cortactin pulls down N WASP. This supports the concept that cortactin binds Tir by means of the N terminus and N WASP via the SH3 domain. Within this case, phosphorylation need to influence only the binding of cortactin to N WASP, in other words, cortactin phosphor ylated on serine would bind each Tir and N WASP whereas cortactin phosphorylated on tyrosine would bind only Tir. Each binding and activation experiments have been also per formed using the Tir phosphorylation mimicking Y474D mutant of Tir. The truth that we did not observe significant variations from WT Tir may perhaps indicates, the mutant will not behave just like the phosphorylated form or the binding and activation of cortactin is inde pendent of Tir phosphorylation on residue 474. Further experiments are necessary to address this question.