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J. Biol. Chem., Vol. 283, Issue 25, 17624-17634, June 20, 2008

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

SongXiao Xu^¶1, Catherine C. L. Wong^||1, Edith H. Y. Tong^**, Stephen S. M. Chung^**, John R. Yates, III^||, YiBing Yin^¶, and Ben C. B. Ko²

From the The State Key Laboratory in Oncology in South China and the Department of Anatomical and Cellular Pathology, Chinese University of Hong Kong, Hong Kong, China, ^¶The Key Laboratory of Laboratory Medical Diagnostics, Ministry of Education, Department of Laboratory Medicine, Chongqing Medical University, Chongqing, China, the ^||Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, and the ^**Department of Physiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China

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 CK11L, 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.

Received for publication, January 11, 2008 , and in revised form, April 11, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grant P41 RR011823. This work was also supported by the Research Grant Council Grants CUHK 7419/03M and 7327/04M, by the University Grant Committee of the Hong Kong Special Administrative Region Area of Excellence Scheme Grant AoE/P-10/01, and by The Chinese University of Hong Kong Direct Grant for Research 2041316 (to B. C. B. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. E-mail: benko{at}cuhk.edu.hk.

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