2,3,7,8-Tetrachlorodibenzo-p-dioxin-Induced Activation of a Protein Tyrosine Kinase, pp60^src, in Murine Hepatic Cytosol Using a Cell-Free System

Abstract

SUMMARY 2,3,7, was found to activate protein kinases under cell-and nucleus-free conditions in isolated C57 mouse liver cytosol (100,000 ϫ g supernatant). This action of TCDD was found to be aryl hydrocarbon receptor (AHR) dependent, concentration dependent, and inhibited by genistein, a tyrosine kinase inhibitor. The lowest concentration of TCDD to produce a statistically significant increase in protein phosphorylation was 10 pM. We also investigated the possibility that a protein kinase is physically associated with the cytosolic AHR complex. Kinase renaturation tests designed to detect reactivated protein kinases after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels revealed the presence of a 60-kDa kinase in the washed immunoprecipitate obtained from liver cytosol using anti-AHR antibody (IgG) and protein A/G/ agarose beads but not when a nonspecific IgG was used instead of anti-AHR antibody. The same 60-kDa band was present in an immunoprecipitate prepared in a similar manner from the same cytosol but with anti-heat shock protein 90 antibody (IgM). This 60-kDa kinase was found to be activated by TCDD treatment of whole cytosol from untreated mice. Moreover, pp60 src immunoprecipitated from cytosol that had been previously treated with TCDD under cell-free conditions exhibited 2-fold more kinase activity than the equivalent preparation treated with a solvent control. Again, such an effect of TCDD could not be detected when a nonspecific IgG was used in place of an anti-pp60 src antibody. Increased protein phosphorylation was observed after direct TCDD treatment of immunoprecipitates obtained using antibodies to AHR and pp60 src , respectively, but not when a nonspecific IgG was used for immunoprecipitation in either case. This observation is consistent with the idea that in cytosol, the AHR and pp60 src coexist as part of a multimeric protein complex that can be specifically coimmunoprecipitated. These results provide evidence that (i) TCDD activates protein kinases in murine hepatic cytosol, (ii) a 60-kDa protein kinase is associated with the cytosolic form of the AHR complex, (iii) ligand binding directly activates this kinase because TCDD treatment of immunoprecipitated AHR complex results in increased protein kinase activity, and (iv) the AHR-associated protein kinase seems to be pp60 src kinase. The current findings provide a clue to a potentially important mechanism by which TCDD can exert rapid, pleiotropic effects through the AHR-associated kinase to alter functions of many proteins through a cascade of protein phosphorylations.

Toxicogenomic investigation of Tetrahymena thermophila exposed to dichlorodiphenyltrichloroethane (DDT), tributyltin (TBT), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)

“…Based on these findings, this model warrants further investigation. The results presented here suggest that Tetrahymena thermophila responds differently when exposed to different types of environmental hormones, and that these responses are similar to those observed in some multicellular organisms [31][32][33][34][35][41][42][43]. Furthermore, interaction network analysis of the exposure to TCDD provided rich information suggesting that T. thermophila would be a good unicellular, eukaryotic model for investigation of molecular mechanisms triggered by exposure to toxicants at the whole-genome level.…”

“…Accordingly, microarray analysis of exposure of Tetrahymena to the toxicants suggested that the functions of the differentially expressed genes varied among toxicant-treated sets. These functions coincided with the characteristics of the toxicants, and some of them have been reported in multicellular organisms [31][32][33][34][35][41][42][43]. Thus, T. thermophila has the potential to be a unicellular model for toxic research because it can reflect general patterns of biological responses to these toxicants that match those of other eukaryotic organisms.…”

“…Function d was common among organisms exposed to oxidative stress [31], which is one of the effects believed to be caused by TCDD [40]. Furthermore, function a is likely related to kinase activity, which is affected by exposure to TCDD [41] and plays an important role in signal regulatory pathways. Additionally, three genes in function c encoded ubiquitin-conjugating enzymes (Appendix Table S6 in the electronic version), which correlates with the ubiquitination reported in response to exposure to TCDD [42,43].…”

Src-mediated aryl hydrocarbon and epidermal growth factor receptor cross talk stimulates colon cancer cell proliferation

“…In rodent adipose and liver tissues, it was reported that Src may be associated with components of the cytosolic AhR complex (5,12). To determine whether AhR and Src are present in the same protein complex in colon epithelial cells, we performed coimmunoprecipitation experiments.…”

The Inhibitory Mechanisms of the Tyrosine Kinase Inhibitors Herbimycin A, Genistein, and Tyrphostin B48 with Regard to the Function of the Aryl Hydrocarbon Receptor in Caco-2 Cells

“…One of the tyrosine kinases that is related to the action of AhR, c-Src, is known to be activated by TCDD in vivo [25][26][27] and in vitro, 28) but it is not clear how c-Src is activated or whether its activity is needed for the activation of AhR. Tyrphostin B48 is known to be a specific inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR), and the EGFR is one of the target molecules that is inhibited by genistein.…”