Aryl Hydrocarbon Receptor (AhR)/AhR Nuclear Translocator (ARNT) Activity Is Unaltered by Phosphorylation of a Periodicity/ARNT/Single-Minded (PAS)-Region Serine Residue
- Department of Veterinary Science and Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, Pennsylvania
Abstract
The aryl hydrocarbon nuclear translocator (ARNT) protein belongs to the family of basic helix-loop-helix (HLH)-periodicity/ARNT/single-minded [Per/ARNT/Sim (PAS)] transcription factors and regulates a range of cellular processes by either homodimerizing or heterodimerizing with other basic HLH-PAS proteins. To date, it has been shown that both the HLH and PAS domains are required for aryl hydrocarbon receptor (AhR) ARNT heterodimerization and that phosphorylation of ARNT is also required for this heterodimerization. Presently, regulation of ARNT with respect to phosphorylation is poorly understood. In an earlier study, murine ARNT was shown to be a phosphoprotein, to display charge heterogeneity, and to have a shift in its predominant isoforms after heterodimerization with the AhR. It was hypothesized that this shift may represent a change in ARNT phosphorylation status. Metabolic [32P]orthophosphate labeling of human ARNT-transfected COS-1 cells, in conjunction with phosphoamino acid analysis, Edman degradation, and phosphopeptide mapping, demonstrated that ARNT is predominantly phosphorylated on serine residues and that serine 348 (S348) in the PAS domain is phosphorylated. Alanine and glutamic acid substitutions were used to demonstrate that loss of phosphorylation at this site did not influence AhR-mediated xenobiotic response elements-driven or ARNT-mediated class B E-box–driven signaling. Additionally, the phosphorylation pattern of ARNT was unaltered after AhR heterodimerization. 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.
Footnotes
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Send reprint requests to: Dr. Gary H. Perdew, Center for Molecular Toxicology, Department of Veterinary Science, Pennsylvania State University, 226 Fenske Lab, University Park, PA 16802. E-mail:ghp2{at}psu.edu
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This work was supported by the National Institute of Environmental Health Science Grants ES04869, ES09272, and ES05863.
- Abbreviations:
- ARNT
- aryl hydrocarbon receptor nuclear translocator protein
- bHLH
- basic helix-loop-helix
- HIF-1α
- hypoxia inducible factor 1α
- PAS
- Per/ARNT/Sim (periodicity/aryl hydrocarbon receptor nuclear translocator/single-minded)
- HLH
- helix-loop-helix
- AhR
- aryl hydrocarbon receptor
- XRE
- xenobiotic response element
- Sim
- single-minded
- TCDD
- 2,3,7,8-tetrachlorodibenzo-p-dioxin
- hAhR
- human aryl hydrocarbon receptor
- hARNT
- human aryl hydrocarbon receptor nuclear translocator
- FBS
- fetal bovine serum
- MEM
- minimum essential medium
- PCR
- polymerase chain reaction
- NP-40
- nonidet P-40
- MENG
- MOPS/EDTA/NaN3/glycerol
- MOPS
- 3-(N-morpholino)propanesulfonic acid
- MENGPI
- MENG containing sodium pyrophosphate, sodium molybdate, sodium fluoride, and sodium vanadate
- DMSO
- dimethyl sulfoxide
- mAb
- monoclonal antibody
- CHAPS
- 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
- PAGE
- polyacrylamide gel electrophoresis
- TLC
- thin-layer chromatography
- CDTA
- trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid
- RLUs
- relative light units
- ANOVA
- analysis of variance
- mARNT
- murine aryl hydrocarbon receptor nuclear translocator
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- Received August 9, 2000.
- Accepted November 20, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
