Phosphorylation of nuclear localization signal inhibits the ligand-dependent nuclear import of aryl hydrocarbon receptor

doi:10.1016/j.bbrc.2004.03.076

Biochemical and Biophysical Research Communications

Volume 317, Issue 2, 30 April 2004, Pages 545-550

https://doi.org/10.1016/j.bbrc.2004.03.076 Get rights and content

Abstract

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor which plays a role as an intracellular mediator of the xenobiotic signaling pathway. We previously identified the minimum nuclear localization signal (NLS) of AhR(13–39): it is composed of two basic amino acid segments, AhR(13–16:RKRR) and AhR(37–39:KRH). In this study, we showed that the two protein kinase C (PKC) sites of Ser-12 and Ser-36 are located one amino acid upstream from each of the two segments, and that a ligand-dependent nuclear import of AhR is inhibited by substitution of aspartic acid for Ser-12 (S12D) or Ser-36 (S36D), which mimics the negative charge of phosphorylation. This observation was supported by microinjection analysis, an in vitro nuclear transport assay, and a luciferase reporter assay, suggesting a two-step mechanism in the ligand-dependent nuclear translocation of AhR.

Section snippets

Materials and methods

Cell culture. Cell lines used for this study were COS7, HeLa, Hepa I, and Madin–Darby bovine kidney (MDBK) cells. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37 °C with 5% CO₂ atmosphere.

Plasmids. cDNA encoding the human AhR was subcloned into the pCMX or SRHis [26] vector for the expression of native or His-tag fused AhR, respectively. Replacement of amino acid residues was performed using a Quick Change site-directed mutagenesis

In vitro phosphorylation of AhR-NLS by protein kinase C

The ligand-dependent nuclear import of AhR is mediated by the bipartite nature of the NLS located in the bHLH domain of the receptor [18], [24]. To elucidate the regulation of the nuclear import of AhR, we examined the effect of phosphorylation–dephosphorylation when it took place close to the NLS of the AhR. Computer-based scanning indicated the existence of three potential PKC phosphorylation sites close to the NLS of amino acid residues 12–42 of the AhR (Fig. 1A). To confirm that

Acknowledgements

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan, Health Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan, and Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency.

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Citation Excerpt :
Regarding transcription, the biochemical state of AHR, ARNT, or CYP1A promoters plus their whole gene body could potentially influence their expression (Helmig et al., 2011; Aluru et al., 2011; Beedanagari et al., 2010). For instance, exposure to environmental contaminants exerts epigenetic regulation through the AHR promoter (i.e., histone modifications, such as the notably decreased trimethylation of histone 3, lysine 27) (Englert et al., 2012) or results in biochemical changes in the AHR protein as a feedback (e.g., ubiquitination and phosphorylation) (Ikuta et al., 2004; Ma and Baldwin, 2000). In addition to transient biochemical changes, in ecology, decades of exposure to PCBs causes selective pressure on certain regions of genes involved in the AHR pathway in wild species, thus having a long-term influence on their expression or function (Wirgin et al., 2011; Reitzel et al., 2014; Reid et al., 2016; Osterberg et al., 2018; Nacci et al., 2016).
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Phosphorylation of nuclear localization signal inhibits the ligand-dependent nuclear import of aryl hydrocarbon receptor

Abstract

Section snippets

Materials and methods

In vitro phosphorylation of AhR-NLS by protein kinase C

Acknowledgements

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

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J. Biol. Chem.

The specific DNA binding activity of the dioxin receptor is modulated by the 90 kd heat shock protein

EMBO J.

A pathway of multi-chaperone interactions common to diverse regulatory proteins: estrogen receptor, Fes tyrosine kinase, heat shock transcription factor Hsf1, and the aryl hydrocarbon receptor

Cell Stress Chaperones