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

23 Citations (Scopus)

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

Fingerprint

Aryl Hydrocarbon Receptors
Energy Transfer
Energy transfer
Chemical activation
Phosphorylation
Assays
Western Blotting
Cells
Epidermal Growth Factor
Environmental Pollutants
1,4-dioxin
CSK tyrosine-protein kinase
Polychlorinated Dibenzodioxins
Tyrosine

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

FRET analysis of protein tyrosine kinase c-Src activation mediated via aryl hydrocarbon receptor. / Dong, Bin; Cheng, Wei; Li, Wen; Zheng, Jie; Wu, Dalei; Matsumura, Fumio; Vogel, Christoph Franz Adam.

In: Biochimica et Biophysica Acta - General Subjects, Vol. 1810, No. 4, 04.2011, p. 427-431.

Research output: Contribution to journalArticle

Dong, Bin ; Cheng, Wei ; Li, Wen ; Zheng, Jie ; Wu, Dalei ; Matsumura, Fumio ; Vogel, Christoph Franz Adam. / FRET analysis of protein tyrosine kinase c-Src activation mediated via aryl hydrocarbon receptor. In: Biochimica et Biophysica Acta - General Subjects. 2011 ; Vol. 1810, No. 4. pp. 427-431.
@article{5470865b52e74bf8811640a533c307f1,
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. General Significance: The FRET assay provides a useful tool to visualize and quantify c-Src kinase activation via AhR in living cells.",
keywords = "AhR, c-Src, COX-2, EGF, FRET, TCDD",
author = "Bin Dong and Wei Cheng and Wen Li and Jie Zheng and Dalei Wu and Fumio Matsumura and Vogel, {Christoph Franz Adam}",
year = "2011",
month = "4",
doi = "10.1016/j.bbagen.2010.11.007",
language = "English (US)",
volume = "1810",
pages = "427--431",
journal = "Biochimica et Biophysica Acta - General Subjects",
issn = "0304-4165",
publisher = "Elsevier",
number = "4",

}

TY - JOUR

T1 - FRET analysis of protein tyrosine kinase c-Src activation mediated via aryl hydrocarbon receptor

AU - Dong, Bin

AU - Cheng, Wei

AU - Li, Wen

AU - Zheng, Jie

AU - Wu, Dalei

AU - Matsumura, Fumio

AU - Vogel, Christoph Franz Adam

PY - 2011/4

Y1 - 2011/4

N2 - 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.

AB - 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.

KW - AhR

KW - c-Src

KW - COX-2

KW - EGF

KW - FRET

KW - TCDD

UR - http://www.scopus.com/inward/record.url?scp=79551618199&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79551618199&partnerID=8YFLogxK

U2 - 10.1016/j.bbagen.2010.11.007

DO - 10.1016/j.bbagen.2010.11.007

M3 - Article

C2 - 21145940

AN - SCOPUS:79551618199

VL - 1810

SP - 427

EP - 431

JO - Biochimica et Biophysica Acta - General Subjects

JF - Biochimica et Biophysica Acta - General Subjects

SN - 0304-4165

IS - 4

ER -