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Title Page
Contents
ABSTRACT 6
ABBREVIATIONS 11
CHAPTER I. GENERAL INTRODUCTION 13
Signal transduction 14
MPK38 14
Trx 19
14-3-3 21
Daxx 23
OBJECTIVES 26
CHAPTER II. Thioredoxin Negatively Regulates Murine Protein Serine-threonine Kinase 38 in a Phosphorylation-dependent Manner 27
ABSTRACT 28
INTRODUCTION 29
MATERIALS AND METHODS 31
Antibodies, plasmids, and Trx small interfering (si) RNAs 31
Cell culture and inducible Trx shRNA cell line 31
Trx mutants 32
MPK38 mutants 33
In vivo and in vitro binding assays 33
MPK38 kinase activity 33
Ubiquitination and luciferase reporter assay 34
Green fluorescent protein (GFP)-based apoptosis assay 34
Statistical analysis 34
RESULTS 35
Trx inhibits MPK38 kinase activity 35
The C-terminal domain of MPK38 is required for Trx binding 37
The interaction between MPK38 and Trx is dependent on redox status 40
Signals that stimulate ASK1, TGF-β, and p53 induce the dissociation of Trx from MPK38-Trx complexes 42
Phosphorylation of Trx on Thr76 by MPK38 is required for the negative regulation of MPK38 kinase activity(이미지참조) 44
Trx inhibits MPK38-stimulated ASK1 signaling in a phosphorylation-dependent manner 46
Trx inhibits MPK38-stimulated TGF-β signaling in a phosphorylation-dependent manner 49
Trx inhibits MPK38-stimulated p53 signaling in a phosphorylation-dependent manner 52
Trx decreases the stability of MPK38 55
Trx-induced dissociation of MPK38 complexes with Smad3, ASK1, or p53 contributes to the negative regulation of MPK38-dependent TGF-β, ASK1, and p53 signaling pathways 59
DISCUSSION 61
CHAPTER III. 14-3-3 Enhances the Activity of Murine Protein Serine-threonine Kinase 38 (MPK38) in a Phosphorylation-dependent Manner 64
ABSTRACT 65
INTRODUCTION 66
MATERIALS AND METHODS 68
Reagents and Antibodies 68
Plasmids 68
Construction of 14-3-3 mutants, 14-3-3(N) and (C) 69
Cell culture and transfection 71
In vivo and in vitro binding assay 71
Recombinant protein purification and in vitro kinase assay 72
Luciferase reporter assay 73
Apoptosis assay 73
RESULTS 74
14-3-3 associates with and stimulates the activity of MPK38 74
MPK38-14-3-3 binding is mediated through their C-terminal domains 78
All 14-3-3 isoforms are phosphorylated by MPK38 81
Association of 14-3-3 with MPK38 was enhanced by stress stimuli for ASK1, TGF-β, and p53 83
14-3-3 stimulates MPK38-mediated ASK1 signaling 85
14-3-3 stimulates MPK38-mediated TGF-β signaling 87
14-3-3 enhances MPK38-induced p53 signaling 89
DISCUSSION 91
CHAPTER IV. Essential role of Daxx Phosphorylation at Thr578 by MPK38 in the Regulation of MPK38 Activity(이미지참조) 93
ABSTRACT 94
INTRODUCTION 95
MATERIALS AND METHODS 97
Materials, antibodies, cell culture, and transfection 97
Plasmid construction 97
In vivo and in vitro binding assay 98
Recombinant proteins purification and in vitro MPK38 kinase assay 99
Luciferase reporter assay 99
RESULTS 100
Identification of Daxx as an interacting partner for MPK38 100
Mapping of the interaction domains of Daxx and MPK38 102
Identification of MPK38 phosphorylation site on Daxx 105
Phosphorylation of Daxx at Thr578 promotes the kinase activity of MPK38(이미지참조) 107
Effects of stress stimuli for ASK1 and TGF-β signaling on the association of Daxx with MPK38 110
Phosphorylation of Daxx at Thr578 is required for the stimulation of MPK38-mediated ASK1 signaling(이미지참조) 112
Daxx phosphorylation at Thr578 is required for the stimulation of MPK38-mediated TGF-β signaling(이미지참조) 114
DISCUSSION 116
SUMMARY 119
REFERENCES 123
초록 139
CURRICULUM VITAE 141
Table 1-1. Mapping of autophosphorylation sites of MPK38 (MELK) by mass spectrometry. 18
Figure 1-1. Domain structure of MPK38, MELK, Xenopus Eg3, and related members of the Snf1/AMPK family. 17
Figure 1-2. Activation of MPK38 signaling pathways based on in vitro and in vitro data. 18
Figure 1-3. Subcellular distribution of thioredoxin and thioredoxin binding proteins. 20
Figure 1-4. 14-3-3 targets implicated in cancer. 22
Figure 1-5. Structural characteristics of Daxx homologs (mouse and human). 25
Figure 1-6. Fas- and TGF-β-mediated apoptotic function of Daxx. 25
Figure 2-1. Inhibition of MPK38 kinase activity by Trx. 36
Figure 2-2. Binding of Trx to MPK38 in vivo and in vitro. 39
Figure 2-3. Redox-dependent interaction between MPK38 and Trx. 41
Figure 2-4. Release of Trx from MPK38-Trx complex by signals that trigger ASK1, TGF-β, and p53 activity. 43
Figure 2-5. Trx phosphorylation on Thr76 by MPK38.(이미지참조) 45
Figure 2-6. Inhibition of MPK38-induced ASK1 downstream signaling by Trx. 48
Figure 2-7. Inhibition of MPK38-induced TGF-β signaling by Trx. 51
Figure 2-8. Inhibition of MPK38-induced p53 signaling by Trx. 54
Figure 2-9. Destabilization of MPK38 by Trx. 58
Figure 2-10. Modulation of complex formation between MPK38 and Smad3 (or Smad7), ASK1, and p53 by Trx. 60
Figure 3-1. 14-3-3 interacts with MPK38 and enhances the kinase activity of MPK38. 77
Figure 3-2. Mapping the interacting domains in MPK38 and 14-3-3. 80
Figure 3-3. MPK38 directly phosporylates 14-3-3 isoforms at specific sites. 82
Figure 3-4. ASK1, TGF-β, and p53 signals potentiate the interaction between 14-3-3 with MPK38. 84
Figure 3-5. 14-3-3 phosphorylation by MPK38 is important for regulating MPK38-mediated ASK1 signaling. 86
Figure 3-6. Phosphorylation of 14-3-3 by MPK38 is necessary for the regulation of MPK38-mediated TGF-β signaling. 88
Figure 3-7. Phosphorylation of 14-3-3 by MPK38 is important for the regulation of MPK38-induced p53 signaling. 90
Figure 4-1. Daxx is an MPK38-interacting protein. 101
Figure 4-2. Mapping of the domains responsible for the interaction between MPK38 and Daxx. 104
Figure 4-3. Identification of potential MPK38 phosphorylation site on Daxx. 106
Figure 4-4. In vitro phosphorylation of Daxx at Thr578 potently enhances MPK38 kinase activity.(이미지참조) 109
Figure 4-5. Stress stimuli for ASK1 and TGF-β signaling induce association of Daxx with MPK38. 111
Figure 4-6. Daxx promotes MPK38-induced AP1-transcriptional activity. 113
Figure 4-7. Daxx enhances MPK38-induced TGF-β-transcriptional activity. 115
Figure 5-1. Schematic representation of the multiple mechanisms regulating the activity and the stability of MPK38. 122
초록보기 더보기
AMPK-related serine—threonine kinase family에 포함되는 MPK38 단백질 인산화효소는 세포사멸을 유도하는 ASK1, TGF-β, p53 신호전달의 다양한 자극들에 의해서 활성화 되어진다. MPK38 신호전달은 줄기세포 재생, 세포성장, 세포주기 조절, 세포사멸 등 많은 세포 내 신호전달 체계에 관여하고 있다. 이 논문에서는 MPK38에 의한 세포사멸 기능에 있어서 중요한 여러 조절인자들을 동정하여 그 특성을 연구하였다. 2 장에서는 MPK38과 물리적으로 결합하여 MPK38 기능을 저해하는 조절인자로써 thioredoxin(Trx)를 분석하였다. MPK38과 Trx mutant 연구를 통하여 Trx cysteine 32와 cysteine 35 잔기는 MPK38의 cysteine 339과 cysteine 377 잔기와 더불어 두 단백질의 결합에 관여하고 있음을 확인하였다. MPK38에 의해 직접적으로 인산화 되는 Trx의 threonine 76 잔기는 MPK38 활성억제, 유비퀴틴화, 분해 등을 조절하는데 있어서 중요한 잔기임을 알 수 있었다. 이러한 결과들로부터 자극이 없는 정상상태에서는 Trx가 threonine 76 잔기 인산화를 통해 MPK38 의존적인 ASK1, TGF-β, p53 세포사멸과정을 억제하는데 관여하고 있음을 관찰하였다. 3 장에서는 14-3-3 단백질들이 MPK38 결합단백질로써의 역할을 수행하며 14-3-3의 C-terminal 부위를 통하여 MPK38과 결합이 이루어지는 것을 보여주었다. 나아가 모든 14-3-3 isoform 들은 MPK38에 의해 각각 θ - Ser156, β - Ser158, r - Ser161, ε - Ser156, ξ — Ser156, ŋ - Ser161, 그리고 σ Ser159 잔기들에 있어서 인산화되며 이러한 인산화 작용은 MPK38 활성을 증가시키는 데 있어서 핵심역할을 수행하고 있음을 알 수 있었다. 4 장에서는 Daxx가 물리적으로 MPK38과 결합하여 MPK38 활성을 증가시키는 것을 보여준다. 한편 MPK38은 Daxx에 의한 MPK38 활성즉진에 있어서 중요한 Daxx threonine 578 부위를 인산화시킨다. 특히, 3 장과 4 장에서는 14-3-3과 Daxx가 인산화 의존적인 방법으로 MPK38 매개 ASK1, TGF-β, p53 신호전달을 촉진시키고 있음을 보여주고 있다. 종합적으로, ASK1, TGF-β, p53과 같은 스트레스 신호의 자극들이 MPK38 억제인자로써 작용하는 Trx를 MPK38로부터 해리시키고 동시에 14-3-3과 Daxx와 같은 MPK38 활성인자들의 MPK38 결합을 유도시킴으로써 MPK38 매개 ASK1, TGF-β, p53 신호전달이 수행되게끔 조절하고 있음을 시사하고 있다. 따라서 이 논문은 MPK38 매개 신호전달과정의 조절 기작을 이해하는데 새로운 시각을 제공할 것이다.
참고문헌 (127건) : 자료제공( 네이버학술정보 )더보기
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