title: Activity of the TonEBP/OREBP transactivation domain varies directly with extracellular NaCl concentration.
abstract: Hypertonicity-induced binding of the transcription factor TonEBP/OREBP to its cognate DNA element, ORE/TonE, is associated with increased transcription of several osmotically regulated genes. Previously, it was found that hypertonicity rapidly causes nuclear translocation and phosphorylation of TonEBP/OREBP and, more slowly, increases TonEBP/OREBP abundance. Also, the C terminus of TonEBP/OREBP was found to contain a transactivation domain (TAD). We have now tested for tonicity dependence of the TAD activity of the 983 C-terminal amino acids of TonEBP/OREBP. HepG2 cells were cotransfected with a reporter construct and one of several TAD expression vector constructs. The reporter construct contained GAL4 DNA binding elements, a minimal promoter, and the Photinus luciferase gene. TAD expression vectors generate chimeras comprised of the GAL4 DNA binding domain fused to (i) the 983 C-terminal amino acids of TonEBP/OREBP, (ii) 17 glutamine residues, (iii) the TAD of c-Jun, or (iv) no TAD. All TAD-containing chimeras were functional at normal extracellular osmolality (300 mosmol/kg), but the activity only of the chimera containing the 983 C-terminal amino acids of TonEBP/OREBP varied with extracellular NaCl concentration, decreasing by >80% at 200 mosmol/kg and increasing 8-fold at 500 mosmol/kg. The chimera containing the 983 C-terminal amino acids of TonEBP/OREBP was constitutively localized to the nucleus and showed tonicity-dependent posttranslational modification consistent with phosphorylation. The activity at 500 mosmol/kg was reduced by herbimycin, a tyrosine kinase inhibitor and by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, a protein kinase CK2 inhibitor. Thus, the 983 C-terminal amino acids of TonEBP/OREBP contain a TAD that is regulated osmotically, apparently by tonicity-dependent phosphorylation.
authors.affiliation: Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. jdf@helix.nih.gov
authors.forename: Joan D
authors.initials: JD
authors.lastname: Ferraris
authors.forename: Chester K
authors.initials: CK
authors.lastname: Williams
authors.forename: Prita
authors.initials: P
authors.lastname: Persaud
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhang
authors.forename: Ye
authors.initials: Y
authors.lastname: Chen
authors.forename: Maurice B
authors.initials: MB
authors.lastname: Burg
mesh_terms: D016227:Benzoquinones; D002460:Cell Line; D002678:Chimera; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D047029:Lactams, Macrocyclic; D050778:NFATC Transcription Factors; D009997:Osmotic Pressure; D017434:Protein Structure, Tertiary; D011809:Quinones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017828:Rifabutin / Q000031:analogs & derivatives; D012965:Sodium Chloride; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
chemical_list: D016227:Benzoquinones; D004268:DNA-Binding Proteins; D047029:Lactams, Macrocyclic; C118125:NFAT5 protein, human; D050778:NFATC Transcription Factors; D011809:Quinones; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D017828:Rifabutin; D012965:Sodium Chloride; C020754:herbimycin