PROTEIN KINASE C REGULATION OF P450 SYSTEM

Wayne State University, Detroit MI

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Abstract

The biological effects of 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and numerous other environmental chemicals are mediated by their binding to the aryl hydrocarbon receptor(AHR), and the induction of a cascade of events including: association of the AHR with the aryl hydrocarbon receptor nuclear translocator (ARNT) protein, binding of the resultant heterodimer to enhancer sequences in target genes, and transcriptional activation of the target genes. One target set consists of several phase I and II biotransformation enzymes commonly referred as the Ah battery. Recent analyses of the expression of select members (Cyp1a1, Cyp1b1, Adhd4, and Nmo1) of the Ah battery in cells transfected with an oncogenic Ha-ras gene, or treated with 12-0-tetradecanoylphorbol-13-acetate (TPA) or microtubulin disrupters have lead us to hypothesize that conditions which modulate the phosphorylation status of AHR/ARNT regulate AHR binding to DNA and its function as a ligand activated transcription factor. In this application, we propose to use in vivo footprinting, a cell-free AHR activating system, and immune co-precipitation protocols to determine if the loss of AHR-DNA complex formation in oncogenic Ha-ras transformed and Colcemid-arrested cells reflects inhibition of AHR/ARNT heterodimerization or heterodimer binding to DNA. Analyses of [32p]orthophosphate labeled AHR/ARNT heterodimerization of heterodimer binding to DNA. Analyses in function correlate with changes in phosphorylation status. An in vitro reconstitution assay will be used to characterize and identify the activity in extracts of Ras and Colcemid-arrested cells that suppress AHR- DNA complex formation. Pharmacological and transfection approaches will be used to determine which signaling pathways activated by oncogenic Ras and Colcemid-arrested cells that suppress AHR-DNA complex formation. Pharmacological and transfection approaches will be used to determine which signaling pathways activated by oncogenic Ras down regulate AHR function. Lastly, immune depletion protocols and transfection analyses with dominant negative transforming growth factor beta (TGF-beta) receptor I or II expression constructs will determine if the transient G/1 block and inhibition of Ah battery induction seen in TPA-treated cells reflect the effects of autocrine TGF-beta production. The studies outlined in this proposal will provide mechanistic information on how AHR function is physiologically regulated in normal tissues, and in cells transformed by an oncogene commonly found in at least 30% (e.g., ras) of all human tumors.