Next we repeated the pedestal formation assay with cells expressi

Next we repeated the pedestal formation assay with cells expressing more info the cort actin S405,418D double mutant, which mimics Erk phos phorylation and activates N WASP in vitro, as well as its non phosphorylatable counterpart. The S405,418D mutant allowed pedestal formation to a simi lar extent as the WT cortactin and to a greater extent, although not significantly greater, than the GFP negative control. The phosphoserine mimicking cortactin mutant accumulated in only 21% of pedestals and showed a weak, diffuse pattern of localization in the cytoplasm and pronounced staining in the nucleus. In contrast, the mutant that abolished Erk phosphorylation impaired pedestal Inhibitors,Modulators,Libraries formation and its own translocation to them. These results suggest that Erk phosphorylation of cortactin contributes to ped estals formation.

Similarly, we wanted to address the role of Src mediated phosphorylation of cortactin. We therefore used the phos photyrosine mimicking mutant Inhibitors,Modulators,Libraries and the phosphotyrosine deficient mutant. In both cases pedestal formation and location of these constructs on them were impaired. These results indicate that Src mediated phoshorylation of cortactin seems to inhibit pedestal for mation and that a dynamic phosphorylation of these tyro sine residues play a role in the formation of pedestals. Total F actin content of cells transfected Inhibitors,Modulators,Libraries with different cortactin mutants Although no appreciable changes in the cellular architec ture were observed, we wanted to exclude the possibility that over expression of cortactin mutants induces a gen eral alteration of the actin cytoskeleton.

Inhibitors,Modulators,Libraries We therefore used flow cytometry to assess the total basal F actin content of the different transfectants. Fig. 1C and 1D show the mean fluorescence of GFP transfectants, which did not significantly differ based on Students t test. As a control, transfected cell were pretreated with Cytochalasin D, a Inhibitors,Modulators,Libraries drug known to inhibit actin polymerization. In addition, this experiment allowed us to calculate the transfection efficiency, which was esti mated as 6070%, based on the analysis of the expression of GFP constructs. EPEC induces N WASP dependent tyrosine phosphorylation of cortactin Contrary to the transient phosphorylation induced by EHEC, EPEC infection of CH7 mouse fibroblasts induces tyrosine phosphorylation of cortactin. Src has been shown to phosphorylate cortactin on tyrosines Y421, 466 and kinase inhibitor Tofacitinib 482, which decreases cortactin affinity for N WASP in vitro. In addition, N WASP deficient cells do not form pedestals. These observations prompted us to examine the phosphorylation status of cortactin in WT mouse embryonic fibroblasts, MEFs deficient in N WASP and MEFs deficient in N WASP in which the pro tein was later restored through retroviral transduction.

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