Regulation of nucleocytoplasmic trafficking of transcription factor OREBP/TonEBP/NFAT5.
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.
Tong
Edith H Y
EH
Department of Chemistry, University of Hong Kong, Hong Kong Special Administrative Region, China.
Guo
Jin-Jun
JJ
Huang
Ai-Long
AL
Liu
Han
H
Hu
Chang-Deng
CD
Chung
Stephen S M
SS
Ko
Ben C B
BC
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007038:Hypotonic Solutions; D007552:Isotonic Solutions; D028884:Karyopherins / Q000502:physiology; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors / Q000235:genetics / Q000378:metabolism*; D049790:Nuclear Export Signals / Q000502:physiology; D019913:Nuclear Localization Signals / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D018160:Receptors, Cytoplasmic and Nuclear / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D000097606:Exportin 1 Protein
D007038:Hypotonic Solutions; D007552:Isotonic Solutions; D028884:Karyopherins; D050778:NFATC Transcription Factors; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D018160:Receptors, Cytoplasmic and Nuclear; D015534:Trans-Activators; D014157:Transcription Factors; D000097606:Exportin 1 Protein; C118125:NFAT5 protein, human