Protein kinase C modulates aryl hydrocarbon receptor nuclear translocator protein-mediated transactivation potential in a dimer context.
Protein kinase C (PKC)- and protein kinase A (PKA)-mediated modulation of the transactivation potential of human aryl hydrocarbon receptor nuclear translocator (hARNT), a basic helix-loop-helix (bHLH)-PAS transcription factor, and the bHLH-ZIP transcription factors USF-1 (for upstream regulatory factor 1) and c-Myc were examined. An 81 nM dose of the PKC activator phorbol-12-myristate-13-acetate (PMA), shown here to specifically activate PKC in COS-1 cells, or a 1 nM dose of the PKA activator 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP) results in 2. 6- and 1.9-fold enhancements, respectively, in hARNT-mediated transactivation of the class B, E-box-driven reporter pMyc3E1bLuc relative to identically transfected, carrier solvent-treated COS-1 cells. In contrast, 81 nM PMA and 1 nM 8-Br-cAMP did not enhance transactivation of pMyc3E1bLuc-driven by USF-1 and c-Myc expression relative to identically transfected, carrier-treated COS-1 cells. Co-transfection of pcDNA3/ARNT-474-Flag, expressing a hARNT carboxyl-terminal transactivation domain deletion, and pMyc3E1bLuc does not result in induction of reporter activity, suggesting PMA's effects do not involve formation of unknown hARNT-protein heterodimers. Additionally, PMA had no effect on hARNT expression relative to Me2SO-treated cells. Metabolic 32P labeling of hARNT in cells treated with carrier solvent or 81 nM PMA demonstrates that PMA does not increase the overall phosphorylation level of hARNT. These results demonstrate, for the first time, that the transactivation potential of ARNT in a dimer context can be specifically modulated by PKC or PKA stimulation and that the bHLH-PAS and bHLH-ZIP transcription factors are differentially regulated by these pathways in COS-1 cells.
Long
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Center for Molecular Toxicology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Chen
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Perdew
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GH
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001483:Base Sequence; D019556:COS Cells; D017931:DNA Primers; D004268:DNA-Binding Proteins; D019281:Dimerization; D004789:Enzyme Activation; D016259:Genes, myc; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D008156:Luciferases / Q000235:genetics; D011493:Protein Kinase C / Q000378:metabolism*; D018336:Receptors, Aryl Hydrocarbon; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects
C497396:ARNT protein, human; D017931:DNA Primers; D004268:DNA-Binding Proteins; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D008156:Luciferases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate