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

BACKGROUND

Activation 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.

METHODS

Here 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.

RESULTS

Western 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%, 5 min after TCDD treatment and continued decreasing until the end of the experiment, 25 min 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.

CONCLUSIONS

The 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 SIGNIFICANCE

The FRET assay provides a useful tool to visualize and quantify c-Src kinase activation via AhR in living cells.