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The role of phosphorylations in hypotonic nuclear export of OREBP/TonEBP/NFAT5
Author(s) -
Ko Ben C.B.,
Xu S.U.,
Wong Catherine C.L.,
Tong Edith H.Y.,
Yates John R,
Chung Stephen S.M.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.933.3
Subject(s) - phosphorylation , serine , microbiology and biotechnology , protein phosphorylation , tonicity , chemistry , alanine , transcription factor , enhancer , biochemistry , biology , protein kinase a , gene , amino acid
The osmotic response element‐binding protein (OREBP), also known as tonicity enhancer‐binding protein (TonEBP) or NFAT5, is hitherto the only known osmo‐sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. Here we showed that nucleocytoplasmic trafficking of OREBP is regulated by Ser155 and Ser158 dual phosphorylation. Alanine scanning mutagenesis revealed that Ser155 is an essential residues in OREBP nucleocytoplasmic trafficking. Tandem mass spectrometry revealed that Ser155, as well as Ser158 of OREBP are phosphorylated in vivo under hypotonic conditions. In vitro phosphorylation assay confirmed the findings from mass spectrometry and further revealed that alanine substitution of Ser155 abolished in vitro phosphorylation of Ser158, whereas an aspartate substitution potentiate the phosphorylation, suggesting that phosphorylation of the two serine residues proceeds in a hierarchical manner in which phosphorylation of Ser155 is a prerequisite for phosphorylation of Ser158. Consistently, alanine substitution of either serine residue prevents nuclear exclusion of OREBP under hypotonic conditions. Our data shed lights into the mechanism that regulates OREBP nucleocytoplasmic trafficking.