LC-MS/MS identification of phosphorylation sites of transcription factor, tonicity-responsive enhancer/osmotic response element-binding protein (TonEBP/OREBP).

Hypertonicity increases the activity of TonEBP, resulting in increased transcription of osmo-protective genes. Hypertonicity also increases phosphorylation of TonEBP and there is indirect evidence that phosphorylation of some specific amino acids may be involved in its activation. However, there has been no direct demonstration of phosphorylation of any specific amino acid. The goal of the present studies was to identify amino acids in TonEBP that are phosphorylated. We used HEK293 cells stably transfected with TonEBP-1-547-V5. We incubated them at 200 or 500 mosmol/kg (NaCl varied) for two hours, extracted nuclear and cytoplasmic proteins, immunoprecipitated TonEBP-V5, subjected the immunoprecipitate to in-solution digestion, enriched for phosphopeptides by immobilized metal affinity chromatography (IMAC), and analyzed peptides from both the eluate and flow-through by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In order to maximize coverage of possible phosphorylation sites, we used 3 different proteolytic enzymes (trypsin, endoproteinase Arg-C, and proteinase K). We find a high probability that several different serines, threonines and tyrosines are phosphorylated in at least one of the conditions. We are mutating the amino acids that we identified to determine their role in TonEBP activity. [ABSTRACT FROM AUTHOR]