PIP5Kγ-dependent PIP2-pool regulates Hippo pathway through Merlin and LATS1
- 주제(키워드) Hippo pathway , Merlin , PIP2 , PIP5K , Hsc70
- 주제(DDC) 570
- 발행기관 아주대학교 일반대학원
- 지도교수 Sang Yoon Lee
- 발행년도 2024
- 학위수여년월 2024. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000033394
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
PIP5Kγ-dependent PIP2-pool regulates Hippo pathway through Merlin and LATS1 The Hippo-Salvador signaling pathway is crucial in the regulation of organ size and development. Despite recent findings linking phos-phatidylinositol (4,5)- bisphosphate (PIP2) and its producing enzyme phosphatidylinositol 4-phosphate 5- kinase (PIP5K) to the Hippo pathway, the underlying mechanism is not well understood. In this study, we have demonstrated the role of PIP5Kγ, a member of the PIP5K family, in activating the Hippo pathway in a PIP2-dependent manner. When we introduced PIP5Kγ87 or PIP5Kγ90, which are the two major splice variants of PIP5Kγ, we observed their activation of large tumor suppressor kinase 1 (LATS1) and their inhibition of Yes-associated protein (YAP). Conversely, when we knocked down PIP5Kγ, the opposite effects were observed. Our research also revealed that PIP5Kγ90 interacts with Merlin's band 4.1/ezrin/radixin/moesin (FERM) domain, forming a complex with PIP2 and LATS1 at the PM. Notably, PIP5Kγ90, but not its kinase-deficient mutant, strengthened the interaction between Merlin and LATS1 and recruited LATS1 to the PM. Moreover, the depletion of PIP5Kγ or the use of its inhibitor (UNC3230) increased colony formation in carcinoma cell lines in a YAP- dependent manner. Additionally, we found that PIP5Kγ90 interacts with heat shock cognate 71-kDa protein (Hsc70), which also plays a role in activating the Hippo pathway. In summary, our findings suggest that PIP5Kγ regulates the Hippo-YAP pathway by forming a functional complex with Merlin and LATS1 at the PIP2-rich PM, and it also interacts with Hsc70, contributing to pathway activation. Keywords: Hippo pathway, Merlin, PIP2, PIP5Kγ, Hsc70
more목차
I. INTRODUCTION 1
A. Hippo pathway 1
B. Merlin and PIP2 3
C. PIP2 and PIP5Kγ 5
D. Hsc70 and PIP5Kγ 7
E. Aim of study 10
II. MATERIALS AND METHOD 11
1. Chemicals and Antibodies 11
2. Expression Constructs 11
3. Cell Culture 12
4. Transfection and Gene Knockdown 12
5. Western blotting (WB) and immunoprecipitation (IP) 13
6. qRT-PCR Analysis 14
7. Immunostaining and Cell Imaging 14
8. Subcellular Fractionation 15
9. Colony Formation and Cell Viability Assays 15
10. Statistical Analysis 16
III. RESULTS 17
1. PIP5Kγ plays a regulatory role in the Hippo-YAP pathway 17
2. Hippo pathway is activated by PIP5Kγ in a PIP2-dependent manner 17
3. PIP5Kγ interacts with Merlin directly through FERM domain and modulating Merlin-LATS1 and YAP-TEAD4 interactions 19
4. The presence of PIP2 generated by PIP5Kγ90 triggers the relocation of LATS1 to regions at the plasma membrane abundant in Merlin 21
5. Merlin and LATS1 are essential in regulating the Hippo-YAP pathway by PIP5Kγ 23
6. PIP5Kγ possesses the potential to inhibit YAP-dependent cell proliferation 23
7. The interaction between PIP5Kγ90 and Hsc70 triggers the activation of the Hippo pathway 25
IV. DISCUSSION 27
V. CONCLUSIONS 32
REFERENCES 33
국문요약 39
