title: Lysine 313 of T-box is crucial for modulation of protein stability, DNA binding, and threonine phosphorylation of T-bet.
abstract: A T-box-containing protein expressed in T cells (T-bet) is a key transcription factor involved in the regulation of Th cell differentiation. Although T-bet-deficient CD4(+) T cells fail to produce IFN-γ and typically differentiate into Th2 cells in vitro, ectopic overexpression of T-bet elevates IFN-γ and suppresses production of IL-2 and Th2 cytokines through different mechanisms. Despite the importance of the T-bet protein level, the regulatory mechanisms that control T-bet protein stability are largely unknown. In this study, we found that T-bet underwent proteasomal degradation via ubiquitination at Lys-313. Despite its robust accumulation following lysine mutation, T-bet(K313R) failed to increase IFN-γ production because of diminished DNA binding activity, as demonstrated in the crystal structure of T-bet-DNA complex. Strikingly, T-bet(K313R) entirely lost the ability to suppress IL-2 production and Th2 cell development; this was due to loss of its interaction with NFAT1. We further identified that the T-bet(K313R) reduced the phosphorylation of T-bet at Thr-302, and that threonine phosphorylation was essential for T-bet interaction with NFAT1 and suppression of NFAT1 activity. Retroviral transduction of T-bet(T302A) into T-bet-deficient cells restored IFN-γ levels compared with those induced by wild-type T-bet, but this mutant failed to inhibit IL-2 and Th2 cytokine production. Collectively, these data show that Lys-313 in the T-box domain is essential for controlling T-bet protein stability via ubiquitin-dependent degradation, T-bet binding to the IFN-γ promoter, and for the interaction with and suppression of NFAT1. Thus, multiple posttranslational modifications of T-bet are involved in fine-tuning cytokine production during Th cell development.
authors.affiliation: College of Pharmacy and Global Top 5 Research Program, Ewha Womans University, Seoul 120-750, South Korea.
authors.forename: Eun Jung
authors.initials: EJ
authors.lastname: Jang
authors.forename: Hye Ryeon
authors.initials: HR
authors.lastname: Park
authors.forename: Jeong-Ho
authors.initials: JH
authors.lastname: Hong
authors.forename: Eun Sook
authors.initials: ES
authors.lastname: Hwang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics; D008239:Lysine / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D050778:NFATC Transcription Factors / Q000378:metabolism; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055550:Protein Stability; D059748:Proteolysis; D016415:Sequence Alignment; D020825:T-Box Domain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018418:Th2 Cells / Q000378:metabolism; D013912:Threonine / Q000378:metabolism*; D014158:Transcription, Genetic; D054875:Ubiquitination; D000098815:T-bet Transcription Factor
chemical_list: D016207:Cytokines; D050778:NFATC Transcription Factors; D020825:T-Box Domain Proteins; D000098815:T-bet Transcription Factor; D013912:Threonine; D007371:Interferon-gamma; D046988:Proteasome Endopeptidase Complex; D008239:Lysine