Abstract
The osmotic response element-binding protein (OREBP), also known as 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. OREBP/TonEBP protein can be detected in both the cytoplasm and nucleus under isotonic conditions, although it accumulates exclusively in the nucleus or cytoplasm when subjected to hypertonic or hypotonic challenges, respectively. Using immunocytochemistry and green fluorescent protein fusions, the protein domains that determine its subcellular localization were identified and characterized. We found that OREBP/TonEBP nuclear import is regulated by a nuclear localization signal. However, under isotonic conditions, nuclear export of OREBP/TonEBP is mediated by a CRM1-dependent, leucine-rich canonical nuclear export sequence (NES) located in the N terminus. Disruption of NES by site-directed mutagenesis yielded a mutant OREBP/TonEBP protein that accumulated in the nucleus under isotonic conditions but remained a target for hypotonicity-induced nuclear export. More importantly, a putative auxiliary export domain distal to the NES was identified. Disruption of the auxiliary export domain alone is sufficient to abolish the nuclear export of OREBP/TonEBP induced by hypotonicity. By using bimolecular fluorescence complementation assay, we showed that CRM1 interacts with OREBP/TonEBP, but not with a mutant protein deficient in NES. Our findings provide insight into how nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by changes in extracellular tonicity.
Highlights
The exposure of mammalian cells to extracellular hypertonicity elicits a genetic program of adaptive cellular responses, which includes the synthesis and accumulation of organic osmolytes such as sorbitol, betaine, and myoinositol [1] to replace intracellular electrolytes that are otherwise deleterious to normal cellular function [2]
osmotic response element-binding protein (OREBP)/tonicity enhancer-binding protein (TonEBP) Undergoes Nucleocytoplasmic Trafficking in Response to Changes in Tonicity, but the C-terminal Transactivation Domain Is Not Required for This Process—OREBP/ TonEBP consists of an N-terminal transactivation domain (AD1, amino acids 1–76) [14], a putative bipartite nuclear localization signal (NLS), a Rel-homologous DNA binding domain that shares significant homology with the NFAT family of transcription factors [10, 11], followed by an extended glutamine-rich transactivation domain at the C terminus, which can be further divided into multiple modulation and activation domains (AD2, amino acids 618 –1476) (Fig. 1A) [14]
We identified a canonical nuclear export sequence (NES) in the extreme N terminus that is primarily responsible for the nuclear export of OREBP/TonEBP under isotonic conditions
Summary
The exposure of mammalian cells to extracellular hypertonicity elicits a genetic program of adaptive cellular responses, which includes the synthesis and accumulation of organic osmolytes such as sorbitol, betaine, and myoinositol [1] to replace intracellular electrolytes that are otherwise deleterious to normal cellular function [2]. Nucleocytoplasmic Trafficking of OREBP/TonEBP/NFAT5 directly showing whether phosphorylation is required for its transcriptional activation activity remain elusive [14] It appears that the proteasome is involved in the hypertonicity-induced nuclear translocation of OREBP/TonEBP, as the prevention of its nuclear accumulation using a proteasome inhibitor blocked hypertonic induction of SMIT and BGT-1 [17]. We identify and characterize three protein motifs, including a nuclear export sequence (NES), a putative auxiliary export domain (AED), and a nuclear localization signal (NLS), that are responsible for OREBP/TonEBP nucleocytoplasmic shuttling in response to changes in extracellular tonicity. These results provide a better understanding of the mechanism of tonicity-regulated nucleocytoplasmic shuttling of OREBP/TonEBP
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