FRET analysis of protein tyrosine kinase c-Src activation mediated via aryl hydrocarbon receptor

Abstract

BackgroundActivation of the protein tyrosine kinase c-Src (c-Src kinase) induced by the exposure to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown in various cell types. Most previous works used Western blot analysis to detect the phosphorylation on the Tyr416 residue, which activates c-Src kinase. MethodsHere we compared the results of c-Src tyrosine phosphorylation via aryl hydrocarbon receptor (AhR)-dependent mechanisms from Western blot analysis with fluorescent resonance energy transfer (FRET) assay detecting c-Src activation after treatment with TCDD to activate AhR in two different human cell types. ResultsWestern blot analyses show time-dependent phosphorylation of c-Src by TCDD in HepG2 and MCF-10A cells. Data from FRET assay visualized and quantified the activation of c-Src kinase induced by TCDD in living cells of both cell types. The FRET efficiency decreased by 20%, 5min after TCDD treatment and continued decreasing until the end of the experiment, 25min after TCDD treatment. PP2, a c-Src specific inhibitor, suppressed both TCDD- and epidermal growth factor- (EGF) induced c-Src activation. In contrast, the AhR antagonist 3′-methoxy-4′nitroflavone (MNF) blocked only TCDD- but not EGF-induced activation of c-Src. ConclusionsThe current study shows that the early activation of c-Src via EGF and AhR signaling pathways can be visualized in living cells using the FRET assay which is in line with Western blot analysis. General SignificanceThe FRET assay provides a useful tool to visualize and quantify c-Src kinase activation via AhR in living cells.