Lysine 313 of T-box Is Crucial for Modulation of Protein Stability, DNA Binding, and Threonine Phosphorylation of T-bet

Abstract

AT-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-gamma and typically differentiate into Th2 cells in vitro, ectopic overexpression of T-bet elevates IFN-gamma 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-gamma 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 Tbet 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-gamma levels compared with those induced by wildtype 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-gamma 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.