2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced activation of a protein tyrosine kinase, pp60src, in murine hepatic cytosol using a cell-free system.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was found to activate protein kinases under cell- and nucleus-free conditions in isolated C57 mouse liver cytosol (100,000 x 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.
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Department of Environmental Toxicology and the Center for Environmental Health Sciences, University of California, Davis, Davis, California 95616, USA.
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D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D000081247:CSK Tyrosine-Protein Kinase; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019833:Genistein / Q000494:pharmacology; D008099:Liver / Q000187:drug effects* / Q000201:enzymology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010761:Phosphorus Radioisotopes; D000072317:Polychlorinated Dibenzodioxins / Q000737:chemistry / Q000494:pharmacology*; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D019061:src-Family Kinases
D001571:Benzoflavones; D010761:Phosphorus Radioisotopes; D000072317:Polychlorinated Dibenzodioxins; D018336:Receptors, Aryl Hydrocarbon; C011512:alpha-naphthoflavone; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases