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

Bin Dong, Wei Cheng, Wen Li, Jie Zheng, Dalei Wu, Fumio Matsumura, Christoph Franz Adam Vogel

Research output: Contribution to journalArticle

24 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)427-431
Number of pages5
JournalBiochimica et Biophysica Acta - General Subjects
Volume1810
Issue number4
DOIs
StatePublished - Apr 2011

Keywords

  • AhR
  • c-Src
  • COX-2
  • EGF
  • FRET
  • TCDD

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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