Title Page
ABSTRACT
Contents
CHAPTER Ⅰ. Regulation of the low-density lipoprotein receptor-related protein LRP6 and its association with disease: Wnt/β-catenin signaling and beyond 14
ABSTRACT 15
1. INTRODUCTION 16
2. MAIN TEXT 19
2.1. Core mechanisms of LRP6 activation via Wnt stimulation 19
2.2. Regulation of LRP6 function and downstream signaling 20
2.3. β-catenin-independent signaling via activation of LRP6 29
2.4. LRP6 Dysregulation and disease 32
3. DISCUSSION 49
4. REFERENCES 50
CHAPTER Ⅱ. LDL receptor-related protein LRP6 senses nutrient levels and regulates Hippo signaling 74
ABSTRACT 75
1. INTRODUCTION 76
2. MATERIAL AND METHODS 80
2.1. Plasmids, siRNAs, and reagents 80
2.2. Cell culture and transfection 80
2.3. Immunoblotting and immunoprecipitation 81
2.4. RNA extraction, cDNA synthesis and quantitative real-time PCR 82
2.5. Luciferase Reporter Assay 83
2.6. Immunofluorescence analysis 84
2.7. Preparation of mouse tissue 84
3. RESULTS 86
3.1. The level of LRP6 is reduced in nutrient starvation 86
3.2. The amount of LRP6 determines the phosphorylation, localization, and transcriptional activity of YAP 88
3.3. Regulation of YAP activity by loss of LRP6 requires Merlin and LATS 90
3.4. To activate Hippo signaling, Merlin switches binding partners from LRP6 to LATS 91
3.5. O-GlcNAcylation on LRP6 is reduced during nutrient starvation and modulation of global O-GlcNAcylation affects the interaction between Merlin and LATS 92
3.6. Nutrient starvation reduces the level of LRP6 and its O-GlcNAcylation in vivo 94
4. DISCUSSION 122
5. REFERENCES 125
CHAPTER Ⅲ. RAI14 links mechanical forces to Hippo signaling 132
ABSTRACT 133
1. INTRODUCTION 134
2. MATERIAL AND METHODS 139
2.1. Plasmids, siRNAs, gRNAs, shRNAs, and reagents 139
2.2. Cell culture and transfection 140
2.3. Lentiviral transduction and generation of genome-edited cells 141
2.4. Lysates preparation, immunoblotting, and immunoprecipitation 141
2.5. RNA extraction, cDNA synthesis and quantitative real-time PCR 143
2.6. Luciferase reporter assay 144
2.7. Immunofluorescence analysis 144
2.8. Proximity-ligation assay 145
2.9. F-actin binding assay 146
2.10. Colony-forming assay 147
2.11. Cell proliferation assay 148
2.12. TCGA analysis 148
2.13. Generation of mouse intestinal and gastric organoids 149
2.14. Statistics and data processing 151
3. RESULTS 152
3.1. RAI14 activates YAP/TAZ 152
3.2. RAI14 interacts with NF2 153
3.3. Regulation of YAP activity via RAI14 is mediated by NF2 155
3.4. Destabilization of F-actin induces RAI14 proteasomal degradation 155
3.5. The interaction between RAI14, NF2, and LATS is regulated by the integrity of F-actin 158
3.6. RAI14 promotes cell proliferation and growth in Hippo signaling-dependent manner 160
4. DISCUSSION 195
5. REFERENCES 199
국문 초록 206
CHAPTER Ⅱ 117
Table 1. List of siRNA sequences 117
Table 2. List of reagents, media and FBS 118
Table 3. List of antibodies 120
Table 4. List of primers 121
CHAPTER Ⅲ 192
Table 1. List of antibodies and materials 192
Table 2. List of siRNA, shRNA, and gRNA 193
Table 3. List of primers 194
CHAPTER Ⅰ 42
Figure 1. Core activation mechanisms of LRP6 via Wnt stimulation. 42
Figure 2. Regulation of LRP6 phosphorylation. 44
Figure 3. Regulation of LRP6 internalization. 45
Figure 4. Regulation of LRP6 maturation & stability. 46
Figure 5. Role of LRP6 as a regulator of other signaling. 47
CHAPTER Ⅱ 97
Figure 1. The level of LRP6 was reduced in nutrient starvation via endocytosis-mediated lysosomal-dependent degradation. 97
Figure 2. LRP6 regulated the phosphorylation, localization and transcriptional activity of YAP. 101
Figure 3. Regulation of YAP phosphorylation and activity by LRP6 requires Merlin and LATS1/2. 105
Figure 4. Merlin changed binding partners from LRP6 to LATS under serum starvation. 107
Figure 5. LRP6 was O-GlcNAcylated and inhibition of O-GlcNAcylation led lysosomal degradation of LRP6. 109
Figure 6. The level of LRP6 O-GlcNAcylation is reduced in nutrient starvation and modulation of global O-GlcNAcylation affected the interaction between... 112
Figure 7. Nutrient starvation reduced the level of LRP6 and its O-GlcNAcylation in vivo. 114
Figure 8. Graphical summary of LRP6-YAP signaling pathway 116
CHAPTER Ⅲ 163
Figure 1. Finding novel regulator of Hippo signaling via TCGA database 163
Figure 2. RAI14 activates YAP/TAZ 166
Figure 3. RAI14 interacts with NF2 171
Figure 4. Regulation of YAP activity via RAI14 is mediated by NF2 174
Figure 5. Destabilization of F-actin induces RAI14 proteasomal degradation 177
Figure 6. RAI14 and NF2 interacts onto F-actin 182
Figure 7. The interaction between RAI14, NF2, and LATS is regulated by the integrity of F-actin 185
Figure 8. RAI14 promotes cell proliferation and growth in Hippo signaling-dependent manner 188
Figure 9. Graphical summary of RAI14-Hippo signaling pathway 190