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.

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.