Okadaic acid increases ARNT homodimer transactivation potential.
The human aryl hydrocarbon nuclear translocator (hARNT) protein belongs to the family of basic helix-loop-helix (bHLH) PAS transcription factors and regulates a range of cellular processes by either homodimerizing or heterodimerizing with other bHLH-PAS proteins. hARNT has been shown to be almost exclusively phosphorylated on serine residues. However, regulation of hARNT with respect to phosphorylation remains poorly understood. The phosphatase inhibitor okadaic acid was used to explore whether a change in hARNT phosphorylation status could influence hARNT homodimer activity. The hARNT homodimer has been shown to bind to E boxes and E-box binding factors are believed to be important in the regulation of cell differentiation and proliferation. Okadaic acid significantly increased hARNT-mediated class B, E-box-driven reporter activity in COS-1 cells, transiently expressing hARNT without affecting hARNT protein levels. This alteration in hARNT-mediated class B, E-box-driven reporter activity correlates with an observed increase in [32P]orthophosphate incorporation into hARNT. Treatment with okadaic acid resulted in a 12-fold increase in [32P]orthophosphate incorporation into hARNT that was transiently expressed in COS-1 cells; an increase in the number of tryptic phosphopeptides generated from hARNT digests on two-dimensional phosphopeptide maps was also observed. Despite the significant increase in [32P]orthophosphate incorporation into hARNT, serine remained the predominantly phosphorylated residue. Clearly, increased serine phosphorylation does not appear to negatively regulate hARNT homodimerization or transactivation potential. These results demonstrate that increased hARNT homodimer signaling in COS-1 cells may result from a direct change in hARNT phosphorylation status.
Levine
S L
SL
Department of Veterinary Science and the Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park 16802, USA.
Perdew
G H
GH
D000818:Animals; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D019556:COS Cells; D004268:DNA-Binding Proteins; D019281:Dimerization; D006801:Humans; D019319:Okadaic Acid / Q000494:pharmacology*; D010449:Peptide Mapping; D010766:Phosphorylation; D011233:Precipitin Tests; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects*; D014357:Trypsin / Q000378:metabolism
C497396:ARNT protein, human; D004268:DNA-Binding Proteins; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D019319:Okadaic Acid; D014357:Trypsin