Lack of an absolute requirement for the native aryl hydrocarbon receptor (AhR) and AhR nuclear translocator transactivation domains in protein kinaseC-mediated modulation of the AhR pathway

Protein kinase C (PKC)-mediated modulation of the aryl hydrocarbon receptor (AhR) pathway was examined in CROK1-derived L10.I cells stably transfected with the pGUDLUC6.1 reporter; pGUDLUC6.1 is solely controlled by four diox in-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-a cetate (PMA), an activator of sn-l,2-diacylglyerol binding PKCs, enhanced t ransactivation of the reporter construct several-fold relative to cells tre ated with a saturating 10 nM TCDD dose alone; this effect was dubbed the "P MA 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 Ah R.ARNT dimer-mediated PMA effect in the L10.I model system; controls demons trate the suitability of the L10.I model for these analyses and that endoge nous AhR and ARNT levels are extremely low in this model. Transient coexpre ssion 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 coexp ression of AhR Delta CVP, a chimeric AhR protein in which the native AhR TA D has been replaced with the VP16 (herpes simplex virus protein IB) TAD (wh ich control experiments demonstrate is unaffected by PMA), and ARNT in L10. I cells indicates that the native AhR TAD is not absolutely required for th is AhR Delta CVP.ARNT dimer to mediate the PMA effect, These observations s trongly suggest that PKC-mediated modulation of the AhR pathway is not abso lutely dependent on coactivators recruited to the AhR.ARNT dimer by the nat ive TADs of the AhR and its heterodimerization partner ARNT. (C) 1999 Acade mic Press.