Phosphorylation By Casein Kinase 1 Regulates Hypotonicity-Induced OREBP/TonEBP Nulcear Exporting

The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or nuclear factor of activated T cell 5 (NFAT5), is hitherto the only known osmo-sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. When the tonicity increases, OREBP translocates from cytoplasm to nuclear, binds to the ORE (Osmotic Response Enhancer) upstream of target genes and then starts or increases their expression. These genes'products, compatible osmolytes, protect cells from deleterious effects of evaluated intracellular electrolyte concentration and restore cells'function. On the other hand, when the tonicity decreases to or below normal level, OREBP moves out of cell nucleus and shuts its target genes down. Osmotic adaptation is particularly important for renal medullary cells, chondrocytes and gastrointestinal epithelia, which physiologically encounter osmolality fluctuationRecently, the mechanism of OREBP nucleocytoplasmic trafficking becomes a hot topic. Although it is well-documented that the subcellular localization and transactivation activity of OREBP/TonEBP are tightly regulated by extracellular tonicity, the molecular mechanisms involved remain elusive. Previously we have identified and characterized three domains of OREBP/TonEBP that regulate its subcellular localization. These domains include a nuclear localization signal (NLS), a canonical nuclear export sequence (NES) and a novel auxiliary export domain (AED). The NES, in conjunction NLS, regulate nucleocytoplasmic shuttling of OREBP/TonEBP under isotonic conditions, whereas AED is indispensable for its nuclear export under both isotonic and hypotonic conditions. NES induced hypotonic nuclear exporting is based on the CRM1, but AED does not. What is the mechanism under the AED induced hypotonic nuclear exporting? As reported, phosphorylation is considered as a crucial factor which could change the protein conformation and protein-protein interaction, and induce protein nuclear export. Have the amino acid residues in AED been phosphorylated under hypotonicity, which can lead the OREBP out of the nuclear? This is the main question we focus in this project.1. We identified the roles of the nine candidate amino acid residues in the AED region using alanine scanning mutagenesis and immuocytochemistry. The result showed the FLAG-OREBP_1-581Δ1-131 S155A mainly located in nuclear under hypotonic treatment, the percentage of fluorescence nuclear localized cells is 66%, while the percentage of the nuclear localized cells of FLAG-OREBP_1-581Δ1-131 is only 9%. This result revealed that S155 was an essential residue that regulated OREBP/TonEBP nucleocytoplasmic trafficking.2. Tandem mass spectrometry revealed that S155, as well as S158 of OREBP/TonEBP were both phosphorylated in living cells under hypotonic conditions. Mutants of FLAG-OREBP_1-581Δ1-131S155A,S158A and S155/158A mainly located in nuclear under hypotonicity, and so did the FLAG-OREBP_1-581Δ1-131S158D and S155/158D, except that the FLAG-OREBP_1-581Δ1-131S155D could still move out of the nuclear under hypotonic treatment. This phenomenon was confirmed by real-time monitoring the hypotonic induced nuclear exporting procedure of GFP OREBP_1-581 and its mutants. We concluded that the sequential phosphorylation of S155 and S158 had the key role in the hypotonic induced nuclear exporting of OREBP.3. The data of in-vitro kinase assay, using the GST-OREBP146-167 and mutants as substrate, showed that the candidate kinase was activated in 20 minutes after the tonicity decreased, and phosphorylation of the two serines proceeded in a hierarchical manner with phosphorylation of S155 priming the phosphorylation of S158, the result of in-vivo gel shift assay was consistent with in-vitro kinase assay. According the information from bioinformatics analysis, we successfully used pharmacological inhibition of Casein Kinase 1 (CK1) to abolish phosphorylation of S158 and impede OREBP/TonEBP nuclear export, and we found that recombinant human CK1 phosphorylated S158,primed by S155 phosphorylation. Knockdown of CK1α1L by siRNA and shRNA, a novel isoform of CK1, inhibited hypotonicity-induced OREBP/TonEBP nuclear export. In summary:These data highlighted that under hypotonicity, an unknown kinase was activated quickly and phosphorylated S155 in the AED of OREBP, sequentially the CK1α1L phosphorylated S158. Based on the dual and sequential phosphorylation of the two serine residues in AED, the OREBP can move out of nuclear to cytoplasm under hypotonicity.