cAMP-Independent Role of PKA in Tonicity-Induced Transactivation of Tonicity-Responsive Enhancer/Osmotic Response Element-Binding Protein

• Published in: Proceedings of the National Academy of Sciences - PNAS 2002-12, Vol.99 (26), p.16800-16805
• Main Authors: Ferraris, Joan D., Persaud, Prita, Williams, Chester K., Chen, Ye, Burg, Maurice B.
• Format: Article
• Language: English
• Subjects: Amino acids; Antibodies; Biological Sciences; Cell Line; Cellular biology; Complementary DNA; Cyclic AMP - physiology; Cyclic AMP-Dependent Protein Kinases - physiology; DNA; DNA-Binding Proteins - physiology; HEK293 cells; Hep G2 cells; Humans; Isoquinolines - pharmacology; Kidneys; NFATC Transcription Factors; Osmotic Pressure; Phosphorylation; Proteins; Sulfonamides; Tonicity; Transactivation; Transcription Factors - physiology; Transcriptional Activation
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.222659799

Hypertonicity-induced increase in activity of the transcription factor tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP) protects renal cells by increasing transcription of genes, including those involved in increased accumulation of organic osmolytes. We previously showed that hypertonicity increases transactivating activity of TonEBP/OREBP. Assay with a binary GAL4 transactivation system showed that the 984 C-terminal amino acids of TonEBP/OREBP (amino acids 548-1531) contain a tonicity-dependent transactivation domain (TAD). Also, amino acids 548-1531 undergo tonicity-dependent phosphorylation, and some inhibitors of protein kinases reduce the tonicity-dependent transactivation. In the present studies we examined the role of protein kinase A (PKA). Results: (i) An inhibitor of PKA (H89) reduces tonicity-dependent increases in transactivation, ORE/TonE reporter activity, and induction of aldose reductase and betaine transporter mRNAs. (ii) Overexpression of the catalytic subunit of PKA (PKAc) increases transactivation activity of amino acids 548-1531 and activity of an ORE/TonE reporter. The increases are much greater under isotonic than under hypertonic conditions. (iii) A dominant-negative PKAc reduces activity of an ORE/TonE reporter. (iv) PKAc activity increases with tonicity but cAMP does not. (v) TonEBP/OREBP and PKAc coimmunoprecipitate. (vi) amino acids 872-1271, including N- and C-terminal polyglutamine stretches, demonstrate tonicity-dependent transactivation, albeit less than amino acids 548-1531, and a similar role for PKA. Conclusions: (i) PKA plays an important role in TonEBP/OREBP activation of tonicity-dependent gene expression; (ii) PKA activation of TonEBP/OREBP appears to be cAMP-independent; and (iii) amino acids 872-1271 are sufficient for tonicity-dependent transactivation of TonEBP/OREBP.