Mutational analysis of the mouse aryl hydrocarbon receptor tyrosine residues necessary for recognition of dioxin response elements.
Tyrosine phosphorylation of the aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix/PER-ARNT-SIM transcription factor family, has been shown to regulate its dioxin response elements (DRE) binding ability, although no specific residues have been directly demonstrated to be phosphorylated. Of the 23 tyrosines in the mouse AhR, 19 are conserved across all mammalian species sequenced thus far. The studies presented here were conducted to examine tyrosine residue(s) that are both likely candidates of phosphorylation and necessary for DNA binding and/or transcriptional activity of the AhR. Two-dimensional gel electrophoresis of phosphatase-treated AhR indicated that the receptor is phosphorylated on serine/threonine and tyrosine residues. Computational analysis predicted several highly conserved tyrosine residues to be phosphorylated. Both the N terminus (amino acids 1-399) and the C terminus (amino acids 399-805) of the mouse receptor synthesized in vitro using a rabbit reticulocyte lysate system are tyrosine phosphorylated as detected by antiphosphotyrosine antibodies. Furthermore, the N-terminal AhR bound DRE in a ligand-dependent manner similar to that by the full-length receptor, suggesting that phosphorylated tyrosines involved in DNA binding are likely located in the region between residues 1 and 399. Mouse AhR tyrosine (Y) residues were evaluated by phenylalanine (F) mutational analysis for both DNA binding (electrophoretic mobility shift assays; EMSAs) and ability to induce a DRE-driven reporter gene in transiently transfected AhR-deficient cells. Of the 12 tyrosine residues in the N-terminal AhR, only a tyrosine 9 mutant (AhRY9F) significantly decreased DRE binding as determined by EMSA. Similarly, only the AhRY9F mutant decreased the DRE-driven luciferase expression in AhR-deficient cells. Overall, these data strongly suggest that the putative posttranslational modification at, or mediated by, tyrosine 9, and not any other individual mouse AhR tyrosine residue, is necessary for AhR DRE binding and transcriptional activity.
Minsavage
Gary D
GD
Department of Environmental Medicine, School of Medicine, University of Rochester, Rochester, NY 14642, USA.
Vorojeikina
Daria P
DP
Gasiewicz
Thomas A
TA
D000818:Animals; D004247:DNA / Q000378:metabolism; D004252:DNA Mutational Analysis; D004147:Dioxins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D015151:Immunoblotting; D051379:Mice; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D018336:Receptors, Aryl Hydrocarbon / Q000737:chemistry* / Q000235:genetics*; D020218:Response Elements; D012984:Software; D014158:Transcription, Genetic; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004147:Dioxins; D018336:Receptors, Aryl Hydrocarbon; D014443:Tyrosine; D004247:DNA