Phosphorylation by Casein Kinase 1 Regulates Tonicity-induced Osmotic Response Element-binding Protein/Tonicity Enhancer-binding Protein Nucleocytoplasmic Trafficking

Songxiao Xu,Catherine C.L Wong,Edith H.Y Tong,Stephen S.M Chung,John R Yates,Yibing Yin,Ben C.B Ko

doi:10.1074/jbc.m800281200

Abstract

The osmotic response element-binding protein (OREBP), also known as tonicity enhancer-binding protein (TonEBP) or NFAT5, is the only known osmo-sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. 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. Here we show that nucleocytoplasmic trafficking of OREBP/TonEBP is regulated by the dual phosphorylation of Ser-155 and Ser-158. Alanine scanning mutagenesis revealed that Ser-155 is an essential residue that regulates OREBP/TonEBP nucleocytoplasmic trafficking. Tandem mass spectrometry revealed that Ser-155 and Ser-158 of OREBP/TonEBP are both phosphorylated in living cells under hypotonic conditions. In vitro phosphorylation assays further suggest that phosphorylation of the two serine residues proceeds in a hierarchical manner with phosphorylation of Ser-155 priming the phosphorylation of Ser-158 and that these phosphorylations are essential for nucleocytoplasmic trafficking of the transcription factor. Finally, we have shown that the pharmacological inhibition of casein kinase 1 (CK1) abolishes the phosphorylation of Ser-158 and impedes OREBP/TonEBP nuclear export and that recombinant CK1 phosphorylates Ser-158. Knockdown of CK1alpha1L, a novel isoform of CK1, inhibits hypotonicity-induced OREBP/TonEBP nuclear export. Together these data highlight the importance of Ser-155 and Ser-158 in the nucleocytoplasmic trafficking of OREBP/TonEBP and indicate that CK1 plays a major role in regulating this process.

Highlights

osmotic response element-binding protein (OREBP)/tonicity enhancer-binding protein (TonEBP) is the only known osmo-sensitive transcription factor in mammalian cells
Ser-155 Located in the auxiliary export domain (AED) of OREBP/TonEBP Plays a Critical Role in Hypotonicity-induced Nuclear Export—Our earlier study showed that the transactivation domain of OREBP/ TonEBP is not required for nucleocytoplasmic trafficking, whereas the nuclear export sequence (NES) and AED are involved in this process (Fig. 1A)
The NES is mainly responsible for the nucleocytoplasmic shuttling of OREBP/TonEBP under isotonic conditions, and the AED plays an indispensable role in the nuclear export of the transcription factor under both isotonic and hypotonic conditions [26]

Summary

EXPERIMENTAL PROCEDURES

Plasmid Constructs and Chemicals—The original OREBP cDNA clone KIAA0827 was a gift from Dr Takahiro Nagase (Kazusa DNA Research Institute, Japan). The pellet was washed with buffer B, resuspended in 100 ␮l of cold buffer C (20 mM HEPES-KOH, pH 7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF), and incubated for 15 min on ice. Cellular debris was removed by centrifugation for 30 min at 13,000 rpm, at 4 °C, and the supernatant was collected as the nuclear extract. In Vitro Phosphorylation Assay—Whole cell, nuclear, or cytoplasm extracts (15 ␮g) obtained from HeLa cells, or recombinant CK1␣1 (Invitrogen), were incubated with GST proteins (10 ␮g) in a kinase buffer containing 20 mM HEPES KOH, pH 7.5, 100 ␮M ATP, 10 mM ␤-glycerol phosphate, 30 mM magnesium chloride, 1 mM DTT, and 2 ␮Ci of [␥-32P]ATP. The primers for detecting CK1␣1L (CSNK1A1L), CK1␣1 (CSNK1A1), CK1-␥3, CK1-⑀, and CK1-␦ were obtained from Qiagen

RESULTS

These data suggested that both

DISCUSSION