Lipid Kinase PIP5Kα Regulates Hippo-YAP Signaling Pathway through Merlin and LATS1
Merlin 및 LATS1을 매개로 하는 지질 인산화효소 PIP5Kα의 Hippo-YAP 신호 전달 경로 조절
- 주제(키워드) PIP5Kα , Merlin , LATS1 , PI（4 , 5）P2 , Hippo pathway
- 주제(DDC) 570
- 발행기관 아주대학교 일반대학원
- 지도교수 Sang Yoon Lee
- 발행년도 2026
- 학위수여년월 2026. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000036204
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
세포 생존능（viability）, 재생, 성장 및 분화를 포함한 핵심적인 세포 프로세스는 조직 항상성의 근본적인 매개체인 Hippo 신호전달 경로에 의해 조절됩니다. 고전적인 MST-LATS 인산화효소 축（kinase axis）은 잘 규명되어 있는 반면, 세포막（PM）에서 이러한 조절 복합체의 모집（recruitment）과 활성화를 촉진하는 상위 메커니즘은 여전히 불분명합니다. 본 연구는 PI（4,5）P2 합성의 주요 효소인 제1형 포스파티딜이노시톨 4-인산 5-인산화효소 알파（PIP5Kα）가 Hippo 경로의 새로운 상위 활성제로서 수행하는 기능적 역할을 조사했습니다. 생화학적 분석, 공초점 현미경 관찰, 기능적 세포 모델을 통해, 우리는 PIP5Kα가 Merlin-LATS1 복합체의 형성과 세포막 모집을 촉진하는 핵심 비계（scaffold）로 작용함을 규명했습니다. 이 과정은 PIP5Kα의 촉매 활성에 엄격하게 의존하며, 활성이 결여된 돌연변이체（kinase-dead mutants）는 유사한 반응을 유도하지 못했습니다. 메커니즘적으로, PIP5Kα에 의해 생성된 PI（4,5）P2는 Merlin이 활성 구조로 전환되도록 유도하며, 이를 통해 LATS1 및 YAP/TAZ의 인산화를 강화하고, 결과적으로 표준 표적 유전자인 CTGF, CYR61, ANKRD1의 발현을 하향 조절합니다. 이 신호전달 축은 세포 밀도 및 혈청 유효성과 같은 세포 외 자극에 의해 역동적으로 조절됩니다. 기능적으로 PIP5Kα 매개 Hippo 활성화는 세포 증식을 억제하여 verteporfin에 의한 YAP 억제의 항증식 효과를 모방하고, 중간엽 줄기세포（MSC）의 지방 생성 분화를 촉진합니다. 특히, FERM 도메인 내의 환자 유래 Merlin 돌연변이는 PIP5Kα-Merlin 인터페이스를 방해하여 종양 억제 신호전달의 상실을 초래합니다. 종합적으로, 우리의 발견은 PIP5Kα가 세포막에서 Hippo 신호전달 경로 구성 성분들의 공간적 조직화를 조절하는 중추적인 항상성 통합자임을 입증합니다. 본 연구는 포스포이노시티드（phosphoinositide） 신호전달과 Merlin-LATS1 축을 연결함으로써 암 진행의 분자적 기반에 대한 새로운 통찰을 제공하고, Hippo 신호전달 경로의 조절 장애를 특징으로 하는 악성 종양에서 PIP5Kα가 잠재적인 치료 표적이 될 수 있음을 강조합니다.
more초록/요약
Critical cellular processes, including cell viability, renewal, growth, and differentiation, are governed by the Hippo signaling pathway, a fundamental mediator of tissue homeostasis. While the classical MST-LATS kinase axis is well-characterized, the upstream mechanisms that promote the recruitment and activation of these regulatory complexes at the plasma membrane （PM） remain incompletely understood. This study investigates the functional role of Type I phosphatidylinositol 4-phosphate 5-kinase alpha （PIP5Kα）, the primary kinase responsible for PI（4,5）P2 synthesis, as a novel upstream activator of the Hippo pathway. Using biochemical assays, confocal microscopy, and functional cell models, we demonstrate that PIP5Kα acts as a critical scaffold to promote the formation and PM recruitment of the Merlin-LATS1 complex. This process is stringently dependent on the catalytic function of PIP5Kα, as kinase-dead mutants fail to elicit a similar response. Mechanistically, PIP5Kα-generated PI（4,5）P2 triggers the transition of Merlin to its active conformation, thereby enhancing LATS1 and YAP/TAZ phosphorylation, which in turn downregulates the canonical target genes CTGF, CYR61, and ANKRD1. This signaling axis is dynamically modulated by extracellular cues such as cell density and serum availability. Functionally, PIP5Kα-mediated Hippo activation inhibits cell proliferation, mirroring the antiproliferative effects of YAP inhibition by verteporfin, and promotes adipogenic differentiation in mesenchymal stem cells （MSCs）. Notably, patient-derived Merlin mutations within the FERM domain disrupt the PIP5Kα- Merlin interface, leading to loss of tumor-suppressive signaling. Collectively, our findings establish PIP5Kα as a pivotal homeostatic integrator that coordinates the spatial organization of Hippo pathway components at the PM. By bridging phosphoinositide signaling with the Merlin-LATS1 axis, this study offers new insights into the molecular basis of cancer progression and highlights PIP5Kα as a potential therapeutic target in malignancies characterized by Hippo pathway dysregulation. Keywords: PIP5Kα, Merlin, LATS1, PI（4,5）P2, Hippo pathway
more목차
Introduction 1
A. Phosphoinositide and cellular signaling 1
1. Phosphoinositide family 1
2. Phosphatidylinositol 4,5-bisphosphate PI（4,5）P2 4
B. Hippo signaling pathway 7
1. The Hippo signaling pathway: overview and mechanism 7
2. The importance of Hippo signaling pathway: cancer and beyond 10
3. Merlin an upstream regulator of Hippo signaling pathway 13
C. PI（4,5）P2 and Hippo signaling pathway 16
Aims of study 18
Materials and methods 19
A. Cell culture and transfection 19
B. Reagents and antibodies 19
C. Expression constructs 20
D. PIP5Kα knockout and gene knockdown 20
E. Lentivirus production and infection 20
F. Merlin point mutations 21
G. Generation of Merlin truncation mutants 22
H. Western blotting （WB） 23
I. Immunoprecipitation （IP） 24
J. Quantitative real-time reverse transcription PCR （qRT-PCR） 24
K. Immunostaining and cell imaging （ICC） 25
L. Measurement of cellular PI（4,5）P2 26
M. GST-fusion protein pull-down assay 26
N. Subcellular fractionation 27
O. Luciferase reporter assay 27
P. Colony formation assay 28
Q. Wound healing assay 28
R. Adipocyte differentiation 28
S. Statistical analysis 28
Results 30
A. The catalytic activity of PIP5Kα is essential for Hippo-YAP/TAZ regulation 30
1. PIP5Kα positively regulates Hippo signaling to suppress YAP/TAZ nuclear activity 30
2. PIP5Kα promotes TAZ degradation via ubiquitination 33
3. PIP5Kα is required to maintain the plasma membrane PI（4,5）P2 pool 36
4. PIP5Kα kinase activity is pivotal for Hippo pathway activation and YAP suppression 39
5. PIP5K isoforms collectively contribute to Hippo-YAP/TAZ activation with pathway-selective effects 42
B. PIP5Kα modulates Hippo signaling in response to cell density and serum stimulation 45
1. PIP5Kα is required for cell density and serum-dependent regulation of Hippo signaling 45
2. Cell density modulates PIP5Kα levels via proteasomal stability and PI（4,5）P2 production 48
C. PIP5Kα promotes the association of LATS1 with Merlin at the plasma membrane via PI（4,5）P2 51
1. PIP5Kα mediates the plasma membrane colocalization of LATS1 and Merlin in a PI（4,5）P2-dependent manner 51
2. PIP5Kα is required for Merlin and LATS1 membrane recruitment 54
D. PIP5Kα regulates the Merlin-LATS1 signaling axis to govern Hippo pathway 57
1. PIP5Kα associates with Merlin and facilitates LATS1 recruitment 57
2. The enzymatic core of PIP5Kα mediates its interaction with Merlin and LATS1 60
3. PIP5Kα directly associates with the F1 subdomain of the Merlin FERM domain 63
4. Merlin acts as a scaffold to bridge PIP5Kα and LATS1 66
5. PIP5Kα-Merlin-LATS1 complex is regulated by Hippo-activating stimuli 69
6. Regulation of the Hippo pathway by PIP5Kα is dependent on Merlin and LATS1 72
E. Biological impact of PIP5Kα-mediated Hippo signaling on cellular processes 75
1. PIP5Kα suppresses cell proliferation in a YAP/TAZ-dependent manner 75
2. PIP5Kα promotes the adipogenic differentiation of mesenchymal stem cells 78
F. Impact of patient-derived Merlin mutations on PIP5Kα binding and Hippo pathway regulation 81
1. Patient-derived Merlin mutations disrupt Hippo pathway activation 81
2. Merlin clinical mutants exhibit differential binding affinity for PIP5Kα 85
3. Merlin-L64P mutation enhances cell proliferation and migratory capacity 88
4. PIP5Kα-mediated suppression of cell growth and migration requires functional Merlin 91
Discussion 94
Conclusion 102
References 103
초록 （Abstract） 118
