Identification and characterization of phosphorylated sites that are required for aryl hydrocarbon receptor (AhR) DNA binding and transactivation

The aryl hydrocarbon receptor (AhR) is a member of the bHLH/PAS transcription factor family to which dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD) binds and mediates various toxic effects. Phosphorylation of the AhR has been suggested to be critical for DNA binding and transcriptional activity, though no sites of phosphorylation have been identified. The studies presented here were conducted to examine AhR residue(s) that are both likely candidates of phosphorylation and necessary for DNA binding and/or transcriptional activity of the AhR. Of the twelve tyrosine residues in the N-terminal DNA binding region of the AhR (residues 1--399), only a tyrosine 9 mutant (AhRY9F) significantly decreased DNA binding and AhR-mediated transcriptional activity. A basic isoelectric point shift was detected by two-dimensional gel electrophoresis of AhRY9F, compared to wild-type. However, an antibody raised to recognize only phosphorylated tyrosine 9 (anti-AhRpY9) confirmed that AhR tyrosine 9 is not a phosphorylated residue required for DNA 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 to AhRY9F, immunopurified full-length wild-type receptor was more rapidly phosphorylated by PKC. Of the 114 serine/threonine residues in the AhR, phosphorylation prediction servers suggested that only 5 of these residues are likely PKC substrates, including both serine 11 and threonine 21. Matrix assisted laser desorption-ionization/time of flight (M&barbelow;ALDI-T&barbelow;OF) m&barbelow;ass s&barbelow;pectrometry (MTMS) revealed that the AhR(4--16) peptide was phosphorylated by PKC and kinase(s) present in rabbit reticulocyte lysate. Furthermore, a mutation of serine 11 to alanine reduced the PKC-elicited phosphate incorporation into the immunopurified full-length AhR. Also, utilizing MTMS analysis of hexahistidine-tagged full-length mouse AhR, we identified several putatively phosphorylated residues. These putative phosphoresidues, including serine 11, were located in the DNA binding region/nuclear localization signal, the HLH and ligand binding/PAS regions as well as the XAP2 interaction/transcription inhibitory domain. Overall, these data implicate a novel role for AhR tyrosine 9 and AhR phosphorylation and demonstrate for the first time that the AhR can be directly phosphorylated by PKC.