Characterization of a novel nuclear import mechanism by nuclear factor of activated T-cells 5 (NFAT5)

Abstract

Nuclear factor of activated T-cells (NFAT5), also known as osmotic response element-binding protein (OREBP) and Tonicity-responsive binding-protein (TonEBP), is a Rel-homology domain containing nuclear factor belongs to the NFAT family transcription factor. NFAT5 is a tonicity-regulated transcription factor, which orchestrates a genetic program that promotes the expression of heat shock proteins and transporters for organic osmolytes for the restoration of cellular homeostasis under hypertonic stress. Among other functions, NFAT5 is best known for its role in the protection of cells in the kidney inner medulla, which are constantly exposed to hypertonic stress due to urine-concentration mechanism. Under hypertonic stress, NFAT5 undergoes nuclear translocation, binds to its cognate DNA enhancer known as the osmotic response element (ORE) of its target genes, and induces gene transcriptions. Nuclear import and export of NFAT5 is essential for its activation and inactivation respectively. Nevertheless, the molecular mechanisms underlying NFAT5 nucleocytoplasmic trafficking remain elusive. Earlier findings suggested that nuclear import of NFAT5 is mediated by a putative novel nuclear import signal (NLS) composed of more than 60 amino acid residues, and requires importin-β as the only nuclear receptor for nuclear import. The aim of my study is to further understand the activities and function of the novel NLS, and to characterize the interaction between the NLS and importin-β. By preparing truncation mutants of the putative NLS and importin-β using bacterial overexpression and purification, I have characterized the interacting domain of the novel NLS with importin-β. Furthermore, using in vitro nuclear import assay, I have defined the sequence requirements and the factors required for nuclear import of NFAT5 for the first time.