Aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) activity is unaltered by phosphorylation of a periodicity/ARNT/single-minded (PAS)-region serine residue

The aryl hydrocarbon nuclear translocator (ARNT) protein belongs to the fam ily of basic helix-loop-helix (HLH)-periodicity/ARNT/single-minded [Per/ARN T/Sim (PAS)] transcription factors and regulates a range of cellular proces ses by either homodimerizing or heterodimerizing with other basic HLH-PAS p roteins. To date, it has been shown that both the HLH and PAS domains are r equired for aryl hydrocarbon receptor (AhR) ARNT heterodimerization and tha t phosphorylation of ARNT is also required for this heterodimerization. Pre sently, regulation of ARNT with respect to phosphorylation is poorly unders tood. In an earlier study, murine ARNT was shown to be a phosphoprotein, to display charge heterogeneity, and to have a shift in its predominant isofo rms after heterodimerization with the AhR. It was hypothesized that this sh ift may represent a change in ARNT phosphorylation status. Metabolic [P-32] orthophosphate labeling of human ARNT-transfected COS-1 cells, in conjunct ion with phosphoamino acid analysis, Edman degradation, and phosphopeptide mapping, demonstrated that ARNT is predominantly phosphorylated on serine r esidues and that serine 348 (S348) in the PAS domain is phosphorylated. Ala nine and glutamic acid substitutions were used to demonstrate that loss of phosphorylation at this site did not influence AhR-mediated xenobiotic resp onse elements-driven or ARNT-mediated class B E-box-driven signaling. Addit ionally, the phosphorylation pattern of ARNT was unaltered after AhR hetero dimerization. Although phosphorylation of S348 did not modulate AhR-ARNT or ARNT-ARNT signaling, phosphorylation of this PAS-region serine residue may be important in other ARNT-mediated gene expression systems.