아주대학교

목차

Introduction 1
PART I. The LKB1-TSSK1B axis inhibits tumor growth through YAP phosphorylation 5
I. Introduction 6
II. Aims of study 9
III. Materials and methods 12
A. Cell culture and transfection 12
B. Virus production and infection 12
C. Western blotting and immuoprecipitation (IP) 12
D. Subcellular fractionation 13
E. Luciferase assay 13
F. Recombinant protein purification 13
G. In vitro kinase assay 14
H. Immunofluorescence staining 14
I. Mutagenesis 15
J. Generation of knockout cell lines 15
K. TA cloning 16
L. shRNA transduction 16
M. Sulforhodamine B (SRB) assy 16
N. Soft agar assay 17
O. Xenograft 17
P. Antibodies and Reagents 17
Q. RNA extraction and quantitative real-time polymerase chain reactions (qRT-PCR) 18
R. Statistical analysis 19
IV. Results 20
1. TSSK1B triggers phosphorylation of YAP, leading to the suppression of its transcriptional activity 20
2. Endogenous TSSK1B modulates YAP phosphorylation and target genes activation 23
3. Endogenous TSSK1B modulates YAP phosphorylation through the Hippo pathway stimulation 26
4. TSSK1B induces YAP phosphorylation through upregulation of LATS1/2 activity via MAP4K4/6/7- and MST1/2-dependent mechanism 29
5. LKB1, the upstream kinase of TSSK1B, induces TSSK1B phosphorylation and mediate subcellular localization 32
6. LKB1-mediated activation of TSSK1B induces phosphorylation of YAP and modifies its subcellular localization 36
7. TSSK1B phosphorylates YAP independent of AMPKα1/2 39
8. TSSK1B directly phosphorylates YAP at Ser94 to regulate its activity 42
9. TSSK1B suppresses tumorigenic potential of YAP without LATS1/2 dependency 45
V. Discussion 50

PART II. GPR54 signaling induces YAP activation through a positive feedback loop in breast cancer 53
I. Introduction 54
II. Aims of study 57
III. Materials and methods 61
A. Cell culture and transfection 61
B. Antibodies and Reagents 61
C. Virus production and transduction 62
D. Western blot and immuoprecipitation (IP) 62
E. In vitro kinase assay 62
F. Luciferase assay 63
G. RNA isolation and quantitative real-time polymerase chain reactions (qRT-PCR) 63
H. Immunofluorescence staining 64
I. Site-directed mutagenesis 64
J. Generation of knockout cell lines using CRISPR/Cas9 system 65
K. TA cloning 65
L. siRNA transfection 66
M. shRNA transduction 66
N. Sulforhodamine B (SRB) assay 66
O. Soft agar assay 67
P. Clonogenic assay 67
Q. Chromatin immunoprecipitation (ChIP-IP) 67
R. Invasion assay 68
S. Migration assay 69
T. Statistical analysis 69
IV. Results 70
1. GPR54 signaling enhances YAP-TEAD transcriptional activity 70
2. Wild-type of GPR54 signaling facilitating YAP activation 73
3. GPR54 signaling enhances YAP activity through Gαq/11 and RhoA activation 76
4. GPR54 signaling inhibits LATS1/2 kinase activity 81
5. GPR54 signaling has a positive feedback loop with the Hippo-YAP pathway 84
6. GPR54 signaling facilitates cancer progression through the Hippo-YAP pathway 88
7. Verteporfin and p234 antagonist regulate YAP activity via GPR54 signaling 91
8. Deletion of YAP attenuates oncogenic potentials by GPR54 signaling 94
V. Discussion 97
Conclusion 100
Reference 102
국문요약 111