Lys-313 of T-bet is crucial for the control of protein stability and suppression of NFAT activity in T cell development

다국어 초록 (Multilingual Abstract)

T-bet, a member of T-box gene family, expressed in T cells is known as a key transcription factor involved in the regulation of Th cell differentiation. While 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 other Th cell cytokines through different mechanisms. Although T-bet protein level is important for transcriptional activities, the molecular mechanisms that control T-bet protein stability are certainly unknown. In this study, these results suggest T-bet underwent proteasomal degradation via ubiquitination at Lys-313. Despite its robust accumulation following lysine mutation, T-betK313R failed to increase IFN-γ production because of a diminished DNA-binding activity, as demonstrated by DNA-pull down assay and the crystal structure of T-bet-DNA complex. Interestingly, T-betK313R completely lost the ability to suppress IL-2 production and Th2 cell development. These results were identified that T-betK313R diminished Thr-302 phosphorylation of T-bet, which was essential site for suppression of NFAT1 activity through T-bet-NFAT1 interaction. Retroviral transduction of T-betT302A into T-bet-deficient CD4+ T cells restored IFN-γ levels compared to those induced by wild-type T-bet, but this mutant failed to inhibit IL-2 and Th2 cytokine production. Taken together, these data indicate that Lys-313 in the T-bet is crucial for regulating T-bet protein stability via ubiquitin-dependent degradation, T-bet binding to IFN-γ promoter, and for the suppression of NFAT1-induced transactivation through interaction. Therefore, multiple post-translational modifications of T-bet are involved in fine-tuning cytokine production during Th cell development.

다국어 초록 (Multilingual Abstract)

T-bet (T-box protein expressed in T cells)은 Th1 세포의 분화와 Th2, Th17, Treg 세포의 분화 조절에 관여하는 중요한 전사인자이다. T-bet 결핍시 CD4+ T cells에서의 IFN- 생성은 감소하는 반면, IL-4, IL-5, IL-13을 분비하는 T helper 2 (Th2) 세포로의 분화는 증가한다. T-bet이 결핍된 T 세포에 T-bet을 재발현 시키면, T-bet을 과발현 시키면, IFN- 생성이 증가하고, IL-2 생성과 Th2 세포로의 분화가 억제 된다. T-bet의 기능은 단백질의 수준에서 중요하게 작용하지만, T-bet의 단백질 안정성에 대한 조절 기전은 알려져 있지 않다. 본 연구에서는 T-bet 단백질이 T cells 에서 proteasome에 의해 분해되며, T-bet Lys-313이 단백질 안정성에 중요한 ubiquitination 부위임을 확인하였다. T-bet Lys-313 변이형(T-betK313R)은 DNA-binding activity가 사라짐으로써 IFN- 생산을 하지 못했다. 이는 DNA와 결합하고 있는 T-bet 단백질 구조로부터 재확인되었다. 또한, T-bet은 NFAT1과의 결합을 통해 IL-2 생성과 Th2세포로의 분화를 억제하는 반면, T-betK313R은 억제하지 못했다. 이는, NFAT1의 의해 생성된 IL-2 생성과 Th2 분화로의 억제 조절에 T-bet Thr-302의 Threonine 인산화가 중요하게 관여함을 확인함으로써 증명하였다. 이러한 기능은 T-bet이 결핍된 쥐에서 분리한 CD4+ T세포에 T-betK313R과 T-betT302A을 재도입하여 T-bet의 기능을 확인하였다. 결과, T-betK313R은 사라진 DNA-binding activity에 의해 IFN- 생산을 하지 못하고, 더불어 IL-2와 Th2 세포의 분화를 저해하지 못하였다. T-betT302A는 IL-2와 Th2 세포 분화의 억제 기능은 사라진 반면, IFN-는 재생산되었다. 종합적으로, 이러한 결과들은 T-bet Lys-313은 T-bet 단백질의 안정성과 IFN- promoter의 결합에 중요하며, T-bet Lys-313은 Thr-302의 Threonine 인산화로 인해 NFAT1과의 상호작용으로 IL-2와 Th2 cytokines을 억제하는데 중요함을 확인하였다. 이러한 결과는, T-bet의 복합적인 단백질 번역 후 변형이 Th 세포의 분화 동안에 cytokine 생성의 조정에 관여함을 증명함으로써 천식이나 면역질환의 치료 개발에 기여 할 수 있는 가능성을 제시하였다.

목차 (Table of Contents)

• Introduction 1
• 1. Structure and functions of T-bet 1
• 2. Transcriptional control of Th cell development 2
• 3. Characterized post-transcriptional modifications of T-bet 3
• 4. In vivo physiological roles of T-bet knockout phenotypes 4
• 5. Regulation of T-bet activity 5
• 6. Specific aims of the study 6
• Material and methods 12
• 1. Reagents 12
• 2. Animals 12
• 3. Cell Culture 12
• 3-1. Isolation and activation of CD4+ T cells 13
• 3-2. In vitro differentiation and retroviral transduction 14
• 4. ELISA 14
• 5. Intracellular Cytokine Staining 15
• 6. Reverse transcription and quantitative real-time PCR analysis 15
• 7. Reporter gene assay 17
• 8. Immunoprecipitation and Immunoblotting 17
• 9. DNA pull-down assay 18
• 10. Nuclear and Cytosolic Fractionations 18
• 11. Immunofluorescence analysis 19
• 12. Statistical analysis 19
• Results 20
• 1. T-bet degradation is blocked by proteasomal inhibitor 20
• 2. T-bet protein undergoes ubiquitin-proteasomal degradation 24
• 3. Lys-313 mutant of T-bet is protected from protein degradation 26
• 4. Lys-313 of T-bet is a potential ubiquitination sit 32
• 5. Lys-313 of T-box domain is essential for DNA binding and transcriptional activity of T-bet 35
• 6. Mutation of K313 abolishes T-bet functions in CD4+ T cell development 40
• 6-1. T-betK313R mutant fails to produce IFN-g. 42
• 6-2. T-betK313R mutant is unable to suppress IL-2 & Th2 cytokine production 44
• 7. Lys-313 is crucial for suppression of IL-2 and Th2 cytokines through interaction with NFAT1 46
• 7-1. T-betK313R is not associated with NFAT1 47
• 7-2. T-betK313R mutant does not suppress NFAT-induced gene promoter suppress 51
• 8. Threonine phosphorylation of T-bet is crucial for regulation through interaction with NFAT1 53
• 8-1. T-betK313R mutant abolishes Threonine phosphorylation of T-bet 53
• 8-2. Thr-302 of T-bet is a threonine phosphorylation site of T-bet 55
• 9. Thr-302 phosphorylation is required for suppression of NFAT activity 57
• 9-1. T-betT302A fails to interact with NFAT1 but interacts with NFkB p65 57
• 9-2. NFAT-mediated gene promoter activity is suppressed by WT but not by TbetT302A 60
• 9-3. Thr-phosphorylation of T-bet is critical for inhibition of NFAT1-mediated IL-2 and Th2 cytokine production 63
• 10. Threonine phosphorylation has no significant effect on IFN-g production by T-bet 66
• 10-1. T-betT302A produces comparable level of IFN-g in T cells 66
• 10-2. T-betT302A mutant sustains DNA-binding activity 68
• 11. Threonine phosphorylation is not required for controlling of T-bet 70
• 12. Lys-313 of T-bet is also essential for the inhibition of NFAT1-mediated IL- 17 expression and Foxp3 activity 72
• Discussion 76
• References 83
• Appendix (publication) 95
• 국문초록 97