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

doi:10.1016/S0006-2952(96)00566-7

Biochemical Pharmacology

Volume 52, Issue 10, 22 November 1996, Pages 1599-1612

https://doi.org/10.1016/S0006-2952(96)00566-7 Get rights and content

Abstract

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 C57B1/6] 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 super-natant 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 pI = 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 μM α-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.

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^☆: This work was supported by ES03575, ES05233, and ES05707 from the National Institute of Environmental Health Sciences, Research Triangle Park, NC.

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Research paperIdentification 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☆

Abstract

Biochem Pharmacol

J Biol Chem

J Biol Chem

J Biol Chem

Reprod Toxicol

Arch Biochem Biophys

Arch Biochem Biophys

J Biol Chem

Biochem Biophys Res Commun

J Biol Chem

Cell

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J Biol Chem

Anal Biochem

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Biochem Pharmacol

Cell

J Biol Chem

Biochem Pharmacol

Research paper
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☆