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Biochim Biophys Acta. 2011 Apr;1810(4):427-31. doi: 10.1016/j.bbagen.2010.11.007. Epub 2010 Dec 9.

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

Author information

1
Center for Health and the Environment, University of California School of Medicine, USA.

Abstract

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.

PMID:
21145940
PMCID:
PMC3049970
DOI:
10.1016/j.bbagen.2010.11.007
[Indexed for MEDLINE]
Free PMC Article

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