Abstract
TonEBP is a Rel domain-containing transcription factor implicated in adaptive immunity, viral replication, and cancer. In the mammalian kidney, TonEBP is a central regulator of water homeostasis. Animals deficient in TonEBP suffer from life-threatening dehydration due to renal water loss. Ambient tonicity (effective osmolality) is the prominent signal for TonEBP in a bidirectional manner; TonEBP activity decreases in hypotonicity, whereas it increases in hypertonicity. Here we found that TonEBP displayed nuclear export in response to hypotonicity and nuclear import in response to hypertonicity. The nuclear export of TonEBP was not mediated by the nuclear export receptor CRM1 or discrete nuclear export signal. In contrast, a dominant nuclear localization signal (NLS) was found in a small region of 16 amino acid residues. When short peptides containing the NLS were fused to constitutively cytoplasmic proteins, the fusion proteins displayed tonicity-dependent nucleocytoplasmic trafficking like TonEBP. Thus, tonicity-dependent activation of the NLS is crucial in the nucleocytoplasmic trafficking of TonEBP. The novel NLS is present only in the vertebrates, indicating that it developed late in evolution.
Highlights
TonEBP, known as NFAT5, is a member of the Rel family of transcriptional activators [1, 2]
We decided to use COS7 cells for this, because we found that COS7 cells, like many other cell lines, had functional TonEBP that was stimulated by ambient hypertonicity
When COS7 cells were switched to hypertonic medium, nuclear localization of TonEBP increased in 30 min, FIGURE 6
Summary
TonEBP (tonicity-responsive enhancer-binding protein), known as NFAT5 (nuclear factor of activated T cell 5), is a member of the Rel family of transcriptional activators [1, 2]. TonEBP is stimulated by hypertonicity (i.e. effective hyperosmolality that causes cell shrinkage due to net water loss across the plasma membrane) [12]. Cytoplasmic shift of TonEBP is observed in the renal medullae of animals suffering from hypokalemia [16] and nephropathy caused by long term treatment with cyclosporine [17]. These reports suggested that a reduction in the tonicity of the renal medullary interstitium signaled for the cytoplasmic shift of TonEBP.
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