Identification of c-Src as the integral component of the cytosolic Ah receptor complex, transducing the signal of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) through the protein phosphorylation pathway

Biochem Pharmacol. 1996 Nov 22;52(10):1599-612. doi: 10.1016/s0006-2952(96)00566-7.


We have shown previously that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) under cell-free conditions causes a significant rise in protein tyrosine kinase activity of cytosol from male guinea pig adipose tissue, and that such an effect of TCDD is Ah-receptor dependent. In the present study, we obtained evidence indicating that c-Src protein kinase is the protein kinase activated by TCDD and that this kinase is associated specifically with the Ah-receptor-complex proteins in cytosol from adipose tissue and liver of guinea pig and liver of C57BI/6J mouse, and in NIH 3T3 mouse fibroblast cells. Here, we present evidence that c-Src protein is functionally attached to the Ah-receptor (AhR) and is specifically activated upon ligand binding. This conclusion is based on several lines of evidence: (a) TCDD caused activation of protein tyrosine kinase activity when administered directly to purified Ah-receptor immunoprecipitate; (b) this stimulatory effect of TCDD was abolished when the cytosol was immunodepleted of c-Src protein or Ah-receptor protein by preincubating with anti-c-Src or anti-Ah-receptor antibody, followed by the addition of TCDD to the remaining portions of cytosol; (c) when Ah-receptor immunoprecipitate was incubated with TCDD, and the kinase(s) released to the supernatant was analyzed on autoradiography of two-dimensional (2D) electrophoresis, 32P-labeled c-Src protein was recognized; (d) the same 32P-labeled-phosphoprotein with M(r) = 60 kDa and pl = 6.1 was found in the immunoprecipitate with anti-c-Src antibody on 2D autoradiograms; (e) this same phosphoprotein disappeared when the supernatant of the Ah-receptor immunoprecipitate was immunodepleted of c-Src protein by anti-c-Src antibody; and (f) a structure-activity relationship study with TCDD and three dioxin-congeners revealed a rank order for their potency in activation of c-Src kinase activity to be identical to that of previously determined toxicity indices: i.e., TCDD > 1,2,3,7,8-pentachlorodibenzo-p-dioxin (1,2,3,7,8-PCDD) > 1,2,4,7,8-pentachlorodibenzo-p-dioxin (1,2,4,7,8-PCDD) > 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD). Consistent with these results, TCDD-induced c-Src kinase activity was abolished when c-Src immunoprecipitate's suspension was preincubated with 0.1 or 1 microM alpha-naphthoflavone (AhR blocker) for 10 min prior to the addition of TCDD. In addition, pretreatment of 3T3 fibroblast cells with 3-methylcholanthrene abolished TCDD-induced c-Src kinase activity in AhR-immunoprecipitate. We conclude that c-Src protein kinase is associated specifically with the AhR complex along with hsp90 in the cytosol of these cells and that upon ligand binding to the Ah-receptor subunit, c-Src is activated and released from the complex.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Adipose Tissue / drug effects
  • Adipose Tissue / metabolism
  • Animals
  • Cytosol / metabolism
  • Guinea Pigs
  • In Vitro Techniques
  • Liver / drug effects
  • Liver / metabolism
  • Male
  • Methylcholanthrene / pharmacology
  • Mice
  • Mice, Inbred C57BL
  • Phosphorylation
  • Polychlorinated Dibenzodioxins / analogs & derivatives
  • Polychlorinated Dibenzodioxins / metabolism
  • Polychlorinated Dibenzodioxins / toxicity*
  • Proto-Oncogene Proteins pp60(c-src) / antagonists & inhibitors
  • Proto-Oncogene Proteins pp60(c-src) / metabolism*
  • Rabbits
  • Receptors, Aryl Hydrocarbon / drug effects*
  • Receptors, Aryl Hydrocarbon / metabolism*
  • Signal Transduction


  • Polychlorinated Dibenzodioxins
  • Receptors, Aryl Hydrocarbon
  • Methylcholanthrene
  • Proto-Oncogene Proteins pp60(c-src)