Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells shed focal adhesion complexes (FAC), a process that includes redistribution of focal adhesion kinase (FAK), -actinin, paxillin, vinculin, F-actin, and spectrin itself. and HT29 cells lose focal adhesion complexes (FAC), a process that includes redistribution of focal adhesion kinase (FAK), -actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments exposed that FAK was tyrosine dephosphorylated, prior to the redistribution of FAK and spectrin. Moreover, phosphatase inhibition clogged cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we display that tyrosine-dephophorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution happen during Pet intoxication. (EAEC) is an growing diarrheagenic pathotype which causes diarrhea in babies and adults, and which has been implicated in epidemic, sporadic, and travelers diarrhea (Nataro, 2005). We have previously reported that EAEC elicits damage to the intestinal mucosa in infected human cells explants, accompanied by exfoliation of enterocytes (Hendersonassays in a number of systems suggest that Pet causes a rise in short circuit current in rat jejunum mounted in Ussing chambers (Navarro-Garciaassays suggest that Pet is able to cleave II-spectrin at a single site in the 11th repeat (Canizalez-Roman & Navarro-Garcia, 2003). But despite several lines of evidence suggesting that spectrin cleavage is definitely a potential mechanism of action of Pet (Villaseca10% SDS-PAGE (6% for spectrin). These are representative results of three independent experiments performed in duplicates. The disassembly of the molecular components of FAC induced by Pet is accompanied by a rapid loss of P-Tyr It is well established that focal adhesion proteins are tyrosine phosphorylated during cell adhesion, and tyrosine dephosphorylated upon detachment from your substratum (Maher, 1993, O’Brien10% SDS- PAGE. (B) Confocal microscopy of semiconfluent monolayers of HEp-2 and HT29 cells treated for 3h with 400nM Pet (c and f), 600nM S260I (b and e) or untreated (a and d). Immunofluorescence labeling was performed to localize phosphotyrosine (P-Tyr) in all panels (green), F-actin (a, b and c) in reddish, and FAK-N (d, e and f) in reddish. Amber corresponds to co-localization of both signals. These are representative results of three independent experiments performed in duplicates. We next investigated if the observed switch in P-Tyr in cells treated with Pet was associated with FAC by immunofluorescence. Unintoxicated adherent HEp-2 and HT29 cells exhibited obvious P-Tyr clusters at focal contacts (Fig. 6-B panels a Cdh1 and d); the appearance was related in cells treated with S260I (Fig. 6-B panels b and e). Unexpectedly, and in contrast with settings, both cell lines intoxicated Istradefylline (KW-6002) with Pet exposed a dramatic switch in the pattern of tyrosine phosphorylation, particularly in the margins of the cytoplasm, where FAC exhibited diminished P-Tyr labeling (Fig. 6-B panels c and f). These data suggest that the time course of FAC disassembly induced by Pet correlates with P-Tyr changes. To characterize these effects further, cell lysates from your cytoskeleton portion were immunoprecipitated using either a monoclonal (Mab) anti-FAK kinase region or polyclonal (Pab) anti-FAK-N. The samples were then immunobloted and probed 1st for P-Tyr and then for FAK, like a control for the immunoprecipitation. These experiments exposed a Istradefylline (KW-6002) dramatic depletion of P-Tyr in native FAK (125kDa) after Pet intoxication in a time dependent manner; regardless of whether the IP was performed using monoclonal or polyclonal antibodies. P-Tyr depletion was also observed among the breakdown products of FAK (at 85kDa). In contrast, these changes in P-Tyr were not observed in unintoxicated settings (Fig. 7-A top panel). Subsequent FAK immunoblots shown that P-Tyr changes described above occurred in FAK itself (Fig. 7-A middle panel). These data present for the first time evidence that Pet induces changes in FAK phosphorylation state. Open in a separate window Open in a separate window Number 7 Pet induces a progressive loss of phosphotyrosine Istradefylline (KW-6002) in immunoprecipitated FAK and does not co-precipitate with either FAK or spectrinThe cytoskeletal portion from HEp-2 cells treated with 400nM of Pet, was immunoprecipitated for either FAK-N (A) or II-spectrin (B). (A) Immunoprecipitation of FAK was performed with either monoclonal antibodies against the kinase region (lanes 1, 2 and 3) or polyclonal antibodies against the N-Terminal region (lanes 4, 5 and 6). Immunoblots from these immunoprecipitations were probed as follows. Top panel, monoclonal antibody against phosphotyrosine; middle panel, polyclonal antibody against FAK-N; bottom panel, polyclonal antibody against Pet. In all three panels, lanes 1 and 4 represent.
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