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A tyrosine kinase-dependent pathway regulates ligand-dependent activation of the dioxin receptor in human
K Gradin1, M L Whitelaw, R Toftgård
1Center for Nutrition and Toxicology, Karolinska Institute, Novum, Huddinge, Sweden.
Abstract:
Signal transduction by dioxin is mediated by the intracellular basic helix-loop-helix dioxin receptor which, in its ligand-activated state, binds to target DNA as a heteromeric complex with the partner factor Arnt. In contrast, the repressed form of the receptor is a complex with hsp90 which appears to maintain the receptor in an inducible conformation. In human keratinocytes dioxin receptor activation has previously been shown to depend on phosphorylation processes. To further dissect mechanisms regulating dioxin receptor function the importance of tyrosine phosphorylation was investigated by the use of specific tyrosine kinase inhibitors. Here we report that the inhibitor genistein inhibited dioxin-dependent induction of expression of the target gene cytochrome P-450IA1. This effect was rapid and reversible and did not lead to altered levels of dioxin receptor protein. Analyses of dioxin receptor or Arnt fusion proteins that function independently of one another showed that the target for genistein action was the dioxin receptor, and, more specifically, a region of the receptor harboring its ligand-binding domain. In addition, function of an unrelated transactivator, the glucocorticoid receptor, was inhibited by genistein while a truncated form lacking the ligand-binding domain was not. A common denominator between the ligand-binding domains of both receptors is their ability to interact with hsp90. Importantly, co-immunoprecipitation experiments showed that genistein inhibited ligand-induced release of hsp90 from the glucocorticoid receptor. Thus, the interaction of these transactivators with hsp90 may be regulated by a tyrosine kinase-dependent pathway.
Insights
Dioxin receptor activation relies on tyrosine phosphorylation. The inhibitor genistein blocks dioxin-induced gene expression by targeting the dioxin receptor’s ligand-binding domain and its interaction with hsp90.
Area of Science:
- Molecular Biology
- Cell Signaling
- Biochemistry
Background:
- Dioxin receptor (AhR) mediates dioxin signal transduction via DNA binding with Arnt.
- The repressed AhR forms a complex with hsp90, maintaining an inducible conformation.
- AhR activation in human keratinocytes is known to involve phosphorylation.
Purpose of the Study:
- To investigate the role of tyrosine phosphorylation in regulating dioxin receptor function.
- To identify the specific molecular targets of tyrosine kinase inhibitors on AhR signaling.
Main Methods:
- Utilized specific tyrosine kinase inhibitors, including genistein.
- Analyzed dioxin-dependent induction of cytochrome P-450IA1 expression.
- Employed fusion proteins to differentiate targets of genistein action.
- Performed co-immunoprecipitation experiments to study receptor-hsp90 interactions.
Main Results:
- Genistein rapidly and reversibly inhibited dioxin-induced cytochrome P-450IA1 expression without altering AhR protein levels.
- Genistein's target was identified as the dioxin receptor, specifically its ligand-binding domain.
- Genistein also inhibited the function of the glucocorticoid receptor, suggesting a common mechanism.
- Genistein interfered with the ligand-induced release of hsp90 from the glucocorticoid receptor.
Conclusions:
- Tyrosine kinase-dependent pathways regulate the interaction of transactivators, like the dioxin receptor and glucocorticoid receptor, with hsp90.
- This pathway is crucial for dioxin receptor activation and subsequent gene expression.