Activity of the TonEBP/OREBP Transactivation Domain Varies Directly with Extracellular NaCl Concentration

• Published in: Proceedings of the National Academy of Sciences - PNAS 2002-01, Vol.99 (2), p.739-744
• Main Authors: Ferraris, Joan D., Williams, Chester K., Persaud, Prita, Zhang, Zheng, Chen, Ye, Burg, Maurice B.
• Format: Article
• Language: English
• Subjects: Amino acids; Benzoquinones; Biological Sciences; Cell Line; Cellular biology; Chimera; Chimeras; Deoxyribonucleic acid; DNA; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Gene Expression - drug effects; Gene expression regulation; Genes, Reporter; HEK293 cells; Hep G2 cells; Humans; hypertonicity; Lactams, Macrocyclic; NFATC Transcription Factors; OREBP protein; Osmosis; Osmotic Pressure; Phosphorylation; Protein Structure, Tertiary; Proteins; Quinones - pharmacology; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Rifabutin - analogs & derivatives; Sodium Chloride; TonEBP protein; Tonicity; Trans-Activators - chemistry; Trans-Activators - genetics; Trans-Activators - metabolism; Transactivation; Transcription Factors - chemistry; Transcription Factors - genetics; Transcription Factors - metabolism; Transfection
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.241637298

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 extra-cellular 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-β-D-ribofuranosyl-benzimidazole, 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.