Lack of an Absolute Requirement for the Native Aryl Hydrocarbon Receptor (AhR) and AhR Nuclear Translocator Transactivation Domains in Protein Kinase C-Mediated Modulation of the AhR Pathway

doi:10.1006/abbi.1999.1452

Archives of Biochemistry and Biophysics

Volume 371, Issue 2, 15 November 1999, Pages 246-259

https://doi.org/10.1006/abbi.1999.1452 Get rights and content

Abstract

Protein kinase C (PKC)-mediated modulation of the aryl hydrocarbon receptor (AhR) pathway was examined in CHOK1-derived L10.I cells stably transfected with the pGUDLUC6.1 reporter; pGUDLUC6.1 is solely controlled by four dioxin-responsive enhancer elements. Co treatment of L10.I cells with 10 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 81 nM phorbol 12-myristate 13-acetate (PMA), an activator of sn-1,2-diacylglyerol binding PKCs, enhanced transactivation of the reporter construct several-fold relative to cells treated with a saturating 10 nM TCDD dose alone; this effect was dubbed the “PMA effect.” A domain swapping and deletional analysis of the native AhR and AhR nuclear translocator (ARNT) protein transactivation domains (TADs) was performed to determine if these domains are absolutely required for the AhR · ARNT dimer-mediated PMA effect in the L10.I model system; controls demonstrate the suitability of the L10.I model for these analyses and that endogenous AhR and ARNT levels are extremely low in this model. Transient coexpression of the AhR and ARNT-474-FLAG, an ARNT protein lacking the native ARNT TAD, in L10.I cells reveals the native ARNT TAD is not absolutely required for the AhR · ARNT-474-FLAG dimer to mediate the PMA effect. Transient coexpression of AhRΔCVP, a chimeric AhR protein in which the native AhR TAD has been replaced with the VP16 (herpes simplex virus protein 16) TAD (which control experiments demonstrate is unaffected by PMA), and ARNT in L10.I cells indicates that the native AhR TAD is not absolutely required for this AhRΔCVP · ARNT dimer to mediate the PMA effect. These observations strongly suggest that PKC-mediated modulation of the AhR pathway is not absolutely dependent on coactivators recruited to the AhR · ARNT dimer by the native TADs of the AhR and its heterodimerization partner ARNT.

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The dioxin (aryl hydrocarbon) receptor as a model for adaptive responses of bHLH/PAS transcription factors
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This review examines the common theme of adaptive responses of bHLH/PAS proteins, using the dioxin receptor as a prototype. The bHLH/PAS family of transcriptional regulators are a group of key developmental and environmental stress sensing proteins. They employ a variety of post-translational control mechanisms to regulate their transcriptional output. Amongst this family, the dioxin receptor is best known for its ability to elicit toxic responses to dioxin and dioxin like chemicals even though it mediates more benign adaptive responses to non-toxic xenobiotics. We discuss what is known about dioxin receptor physiology, both adaptive and inherent, along with its molecular regulation and put this into the context of the wider bHLH/PAS family. We also raise the issue of its toxic responses, in particular the idea that it is the dysregulation of its poorly characterised housekeeping functions that leads to these outcomes.
Phenylthiourea as a weak activator of aryl hydrocarbon receptor inhibiting 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced CYP1A1 transcription in zebrafish embryo
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Therefore, protein kinase C (PKC) might exert its effects by increasing the transactivation potential of the AHR/ARNT heterodimer [48]. In this manner, PKC is thought to increase the ability of coactivator proteins to interact with AHR/ARNT complex [52,53]. A number of nuclear receptor coactivators were shown to interact with AHR, such as ERAP140 [54], RIP140 [55], p300, CBP [56], BRG-1 [57] and the three members of the p160 family of coactivators: NcoA1 (SRC1), NcoA2 (GRIP-1 and TIF-2) and NcoA3 (AIB-1, p/CIP, and ACTR) [58,59].
The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that can be activated by a diverse synthetic and naturally-occurring chemicals, such as the halogenated aromatic hydrocarbons (HAHs) and the non-halogenated polycyclic aromatic hydrocarbons (PAHs). The liganded AHR modulates the genetic activity of a variety of xenobiotic-responsive genes, including cytochrome P4501A1 (CYP1A1). The tyrosinase inhibitor 1-phenyl-2-thiourea (PTU) is widely used in zebrafish research to suppress pigmentation in developing embryos/fry. Here we showed that 0.2 mM PTU induced a basal level of CYP1A1 transcription in zebrafish embryonic integument as early as 24 h postfertilization (hpf) stage. Subsequently, PTU induced CYP1A1 transcription in blood vessels at 36 hpf. During larval stage, the liver and all pharyngeal arch vessels of PTU-treated embryos exhibited CYP1A1 transcription as well. Comparing to TCDD, PTU induces CYP1A1 transcription with much lower efficacy in zebrafish embryos. Coincubating the embryos with PTU and TCDD led to repressing TCDD-induced CYP1A1 transcription. Mechanistic studies indicated that both of PTU- and TCDD-mediated CYP1A1 transcriptions are modulated by the same AHR–ARNTsignaling pathway.
The aryl hydrocarbon receptor (AhR) tyrosine 9, a residue that is essential for AhR DNA binding activity, is not a phosphoresidue but augments AhR phosphorylation
2004, Journal of Biological Chemistry
Citation Excerpt :
Immunopurified AhR Is Phosphorylated by PKC, and AhRY9F Inhibits PKC-elicited Phosphorylation—The inability of the anti-AhRpY9 antibody to recognize the AhR together with the two-dimensional gel electrophoresis data suggesting that a mutation of tyrosine 9 still results in a less negatively charged receptor support a hypothesis that tyrosine 9 is required for normal post-translational modification of other AhR residues. There is much published data implicating the importance of phosphorylation for AhR activity and, in particular, PKC has been observed to be important in the regulation of AhR activity in whole cells (for example, Refs. 26 and 29–32). In an attempt to determine if PKC was capable of phosphorylating the full-length AhR, wild-type and mutant AhRs were immunoprecipitated from rabbit reticulocyte lysate and incubated with purified PKC and [32P]ATP.
We delineate a mechanism by which dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD)-mediated formation of the aryl hydrocarbon receptor (AhR) DNA binding complex is disrupted by a single mutation at the conserved AhR tyrosine 9. Replacement of tyrosine 9 with the structurally conservative phenylalanine (AhRY9F) abolished binding to dioxin response element (DRE) D, E, and A and abrogated DRE-driven gene induction mediated by the AhR with no effect on TCDD binding, TCDD-induced nuclear localization, or ARNT heterodimerization. The speculated role for phosphorylation at tyrosine 9 was also examined. Anti-phosphotyrosine immunoblotting could not detect a major difference between the AhRY9F mutant and wild-type AhR, but a basic isoelectric point shift was detected by two-dimensional gel electrophoresis of AhRY9F. However, an antibody raised to recognize only phosphorylated tyrosine 9 (anti-AhRpY9) confirmed that AhR tyrosine 9 is not a phosphorylated residue required for DRE binding. Kinase assays using synthetic peptides corresponding to the wild-type and mutant AhR residues 1–23 demonstrated that a tyrosine at position 9 is important for substrate recognition at serine(s)/threonine(s) within this sequence by purified protein kinase C (PKC). Also, compared with AhRY9F, immunopurified full-length wild-type receptor was more rapidly phosphorylated by PKC. Furthermore, co-treatment of AhR-deficient cells that expressed AhRY9F and a DRE-driven luciferase construct with phorbol 12-myristate 13-acetate and TCDD resulted in a 30% increase in luciferase activity compared with AhRY9F treated with TCDD alone. Overall, AhR tyrosine 9, which is not a phosphorylated residue itself but is required for DNA binding, appears to play a crucial role in AhR activity by permitting proper phosphorylation of the AhR.
Protein kinase C δ is activated in mouse ovarian surface epithelial cancer cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
2004, Toxicology
Interactions between the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and protein kinase C (PKC) signaling pathways are governed in cell and tissue-specific manners, albeit the physiological significance of which is unclear. This research sought to define the effects of TCDD on the PKC pathway using a mouse ovarian surface epithelial cancer cell line (ID8). Phorbol-12-myristate-13-acetate (PMA) potentiated (1 nM) TCDD-induced 7-ethoxyresorufin-O-deethylase (EROD) activity after 24 h of treatment, and pre-treatment with (1 μM) of either a general PKC inhibitor (BisI) or PKCδ-specific inhibitor (Rotterlin) abolished the potentiation indicating that activation of PKC enhances TCDD signal transduction. Western blot analysis revealed that unstimulated ID8 cells express PKCα, β, ε, τ, λ and RACK1. PKCγ, η, θ and DGKθ were not detected. TCDD (1 nM) increased PKCδ protein approximately eight-fold after 24 h of treatment and this effect was dose-dependent (0.1–100 nM); other PKC isoforms and related signaling proteins tested were unaffected by TCDD treatment. Immunofluorescent microscopy revealed that TCDD (1 nM) promoted the subcellular redistribution of PKCδ, from the cytoplasm and the nucleus to the perinuclear area after 2 h of treatment, however, after 24 h of treatment PKCδ was observed in nuclear structures that resembled nucleoli. TCDD (1 nM) also increased total PKC and PKCδ-specific kinase activities in biphasic time-responsive manners. Total PKC and PKCδ-specific activities increased after 1–2 h of treatment. Then TCDD increased the total PKC activity again after 12 h of treatment, whereas, PKCδ-specific activity resurged at 24 h and remained elevated at 48 h after treatment. The results indicate that TCDD preferentially induces PKCδ protein expression and phosphotransferase activity, and its membrane translocation, indicating a potential intracellular role for PKCδ as an effector molecule for TCDD-mediated biological events in this ovarian cancer cell line.
Proteasome inhibition induces nuclear translocation of the dioxin receptor through an Sp1 and protein kinase C-dependent pathway
2003, Journal of Molecular Biology
The dioxin receptor (AhR), in addition to its role in xenobiotic-induced carcinogenesis, appears to participate in cell proliferation, differentiation and organ homeostasis. Understanding potential mechanisms of activation of this receptor in the absence of exogenous ligands is therefore important to study its contribution to endogenous cellular functions. Using mouse embryo primary fibroblasts, we have previously shown that proteasome inhibition increased AhR transcriptional activity in the absence of xenobiotics. We suggested that proteasome inhibition-dependent AhR activation could involve an increase in the expression of the partner protein dioxin receptor nuclear translocator (ARNT). Since ARNT over-expression induced nuclear translocation of the AhR, and ARNT-deficient cells were unable to translocate this receptor to the nucleus upon proteasome inhibition, we have analyzed the effect of proteasome inhibition on the expression of regulatory proteins controlling ARNT levels. Treatment with the proteasome inhibitor MG132 increased endogenous Sp1 phosphorylation and its DNA-binding activity to the ARNT promoter. Sp1 phosphorylation and binding to the ARNT promoter, ARNT over-expression and AhR nuclear translocation were inhibited by GF109203X, a protein kinase C-specific inhibitor. In addition, MG132 stimulated protein kinase C activity in MEF cells with a pattern similar to that observed for ARNT expression. These data suggest that cellular control of protein kinase C activity, through Sp1 and ARNT, could regulate AhR transcriptional activity in the absence of xenobiotics.
DNA binding and protein interactions of the AHR/ARNT heterodimer that facilitate gene activation
2002, Chemico-Biological Interactions
Gene activation by the aryl hydrocarbon receptor (AHR) and its DNA binding partner, the aryl hydrocarbon receptor nuclear translocator (ARNT) requires a number of sequential steps that occur following the binding of ligand and entry of the AHR into the nuclear compartment. This includes heterodimerization of the AHR and ARNT, formation of the appropriate amino acid/nucleotide contacts at the GCGTG recognition site and interactions between either the AHR or ARNT with proteins that facilitate changes in chromatin structure. The majority of these steps are likely modulated by changes in both phosphorylation and oxidation status of the AHR, ARNT and associated proteins. Studies of both the basic helix-loop-helix transcription factors and the nuclear hormone receptor family can provide significant insights into how this unique signaling pathway activates its target genes.

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¹: To whom correspondence should be addressed at Center for Molecular Toxicology, Department of Veterinary Science, Pennsylvania State University, 115 Henning Building, University Park, PA 16802. Fax: (814) 863-6140. E-mail: [email protected].

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Regular ArticleLack of an Absolute Requirement for the Native Aryl Hydrocarbon Receptor (AhR) and AhR Nuclear Translocator Transactivation Domains in Protein Kinase C-Mediated Modulation of the AhR Pathway

Abstract

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Regular Article
Lack of an Absolute Requirement for the Native Aryl Hydrocarbon Receptor (AhR) and AhR Nuclear Translocator Transactivation Domains in Protein Kinase C-Mediated Modulation of the AhR Pathway