title: FRET analysis of protein tyrosine kinase c-Src activation mediated via aryl hydrocarbon receptor.
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.
  
  
  CONCLUSIONS
  The FRET assay provides a useful tool to visualize and quantify c-Src kinase activation via AhR in living cells.
authors.affiliation: Center for Health and the Environment, University of California School of Medicine, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Dong
authors.forename: Wei
authors.initials: W
authors.lastname: Cheng
authors.forename: Wen
authors.initials: W
authors.lastname: Li
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
authors.forename: Dalei
authors.initials: D
authors.lastname: Wu
authors.forename: Fumio
authors.initials: F
authors.lastname: Matsumura
authors.forename: Christoph Franz Adam
authors.initials: CF
authors.lastname: Vogel
mesh_terms: D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D004785:Environmental Pollutants / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D019061:src-Family Kinases
chemical_list: D004785:Environmental Pollutants; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human