This strategy was vital since the pellets didn’t contain simply detectable levels of N WASP. As previously described, the SH3 domain of cortactin was in a position 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 area of cortactin is involved in binding Tir, when the SH3 domain is involved in binding N WASP. Discussion Cortactin is really a scaffold protein implicated in quite a few cellular processes due to the fact it directly contributes to cytoskeleton remodeling. Cortactin also has oncogenic properties due to its part in controlling invadopodia formation and cell migration. Additionally, cortactin has emerged as an impor tant target of several pathogens, such as enteropath ogenic E. coli that manipulate the actin cytoskeleton so that you can invade the host and propagate there.
EPEC cause serious diarrheal illness in humans by colonizing the gut mucosa and creating A E lesions. EPEC attach to mammalian intestinal cells and induce reorganization of your actin cytoskeleton into pedestal inhibitor kinase inhibitors like structures below neath the bacteria. A critical event for pedestal formation could be the insertion in to the host cell membranes with the EPEC effector Tir, which is initially injected in to the cell by a kind III secretion technique. Tir mimics signaling pathways from the infected cell. Therefore it can serve as a effective model sys tem to study eukaryotic transmembrane signaling. In truth, the Tir Nck N WASP pathway is the principal one through which actin polymerizes in EPEC pedestals. Those motives prompted us to study cortactin signaling for the duration of EPEC infection utilizing N WASP deficient cells.
Even though cortactin localizes to pedestals and its truncated types exert a dominant negative effect, its function is just not clear. As an example, does cortactin on its own contribute to actin polymerization in pedestals Our transfection exper iments with in the know the GFP W22A cortactin point mutant dem onstrate that cortactin binding and activation of your Arp2 three complicated is necessary for pedestal formation, which sug gests that cortactin certainly contributes to effective actin polymerization. A complementary study used a comparable strategy to examine the part of cortactin domains on pedestal formation. It reported identical results to ours with regards to WT cortactin plus the mutant W525K. On the other hand, the W22A mutant was not studied in that function. To address the part of Erk and Src phosphorylation of cort actin, we employed both phosphorylation mimicking and non phosphorylatable mutants, previous research have utilized only the former. Therefore, we have been able to detect a neutral effect on pedestal formation of mutant that mimics phosphorylation by Erk, while the Erk non phosphorylatable form blocked pedestal formation.