Thus, we conclude that phosphorylation of cortactin by Erk may well positively regulate pedestal formation. Our conclusion can also be supported by other studies, over expression of a mutant of cortactin mimicking phosphorylation on serine enhanced invadopodia formation in cells in which endog enous cortactin expression had previously been decreased by siRNA. We could not use a equivalent strategy inside the present study since the cells detached and died upon EPEC infection. The presence of endog enous cortactin might clarify why the SD mutant did not cause considerably much more pedestals than WT, despite the fact that an increase was detectable. Experiments with cortactin defi cient cells may possibly supply the definitive answer to this ques tion.
In contrast, phosphoserine mimicking cortactin accumulated in only 1 fourth of pedestals and showed weak full report diffuse staining within the cytoplasm in addition to a strong nuclear staining. We don’t comprehend this distribution, and we are at the moment investigating it. Src phosphorylates cortactin on positions Y421, 466, and 482. Hence we employed phosphorylation mimicking and non phosphorylatable triple mutants. In both cases pedestal formation was impaired, also because the accumu lation with the mutant proteins towards the pedestals that did form. These outcomes indicate that phoshorylation of cortac tin by Src inhibits pedestal formation. Exactly the same conclu sion was reached utilizing the double Y421,466D mutant which partially mimics Src phosphorylation, which further supports the concept that cortactin phosphorylated on tyrosine inhibits pedestal formation.
The truth that both Src mimicking and non phosphorylatable cortactin types inhibited the formation of pedestals could possibly indicate that a dynamic phosphorylation of those tyrosine residues play a role in the formation of pedestals. Lastly, we can exclude that the effects on pedestals had been as a consequence of alterations within the total actin content hop over to this website from the transfectants, since the content was similar for all transfectants exam ined. This argues that our final results on pedestal for mation reflect the certain effects of phosphorylation or lack of phosphorylation. A crucial locating of this study is the fact that tyrosine phosphor ylation of cortactin is abrogated in N WASP deficient cells but recovered by N WASP re expression. In agree ment with these benefits, preliminary data employing an anti physique against cortactin phosphorylated on serine 405 show that EPEC induces serine phosphorylation of cortac tin, which is not up regulated in EPEC infected N WASP deficient cells. Importantly, the lack of cortac tin tyrosine phosphorylation was not resulting from a defect on Src activation. We believe that only the fraction of cortactin which has translocated to the pedestals is offered for serine and tyrosine phosphorylation.