Identification of novel nuclear export domains of transcription factor OREBP/TonEBP/NFAT5

Abstract

The Osmotic Response Element-Binding Protein (OREBP), also known as the Tonicity Enhancer-Binding Protein (TonEBP) or NFAT5, regulates the hypertonicity-induced expression of a battery of genes crucial for the adaptation of mammalian cells to extracellular hypertonic stress. The activity of OREBP/TonEBP is regulated at multiple levels including nucleocytoplasmic trafficking. Specifically, OREBP/TonEBP undergoes nuclear translocation and export under hypertonic and hypotonic condition respectively. Here we show that, by immunocytochemistry and GFP fusion, the transactivation domain of OREBP/TonEBP is dispensable for proper subcellular localization of OREBP/TonEBP in response to change in osmolality. Under isotonicity, OREBP/TonEBP undergoes nucleocytoplasmic shuttling which can be blocked by leptomycin B (LMB) treatment. Unexpectedly, two leucine-rich motifs that exhibit significant sequence similarity to canonical nuclear export signal (NES) located in the N-terminal of OREBP/TonEBP are not responsible for the export. Nevertheless, we identified a CRM1 responsive protein domain (designated NES-A) responsible for the nucleocytoplasmic shuttling of OREBP/TonEBP under isotonicity. Disruption of NES-A increases nuclear translocation of OREBP/TonEBP, yet hypotonicity is able to direct nuclear export of this mutant. Moreover, we identified a second protein motif (designated NES-B) that is critical for OREBP/TonEBP nuclear export under both isotonic and hypotonic conditions. Disruption of the NES-B alone is sufficient to cause aberrant nuclear translocation of OREBP/TonEBP irrespective of extracellular tonicity. Our study provides novel insights on the regulation of nucleo-cytoplasmic trafficking of OREBP/TonEBP.