cAMP-independent role of PKA in tonicity-induced transactivation of tonicity-responsive enhancer/ osmotic response element-binding protein.
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.
Ferraris
Joan D
JD
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Department of Health and Human Services, Bethesda, MD 20892, USA. jdf@helix.nih.gov
Persaud
Prita
P
Williams
Chester K
CK
Chen
Ye
Y
Burg
Maurice B
MB
D002460:Cell Line; D000242:Cyclic AMP / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D050778:NFATC Transcription Factors; D009997:Osmotic Pressure; D013449:Sulfonamides; D014157:Transcription Factors / Q000502:physiology*; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D007546:Isoquinolines; D050778:NFATC Transcription Factors; D013449:Sulfonamides; D014157:Transcription Factors; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide