PO.MCB03.03 · 分子与细胞生物学
定义调控Hippo信号输出的DLG5-NUAK2-LATS1复合物
Defining a DLG5-NUAK2-LATS1 complex that regulates Hippo signaling output
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
引言：Hippo信号通路是细胞生长和器官大小的关键调控因子，其失调导致转录效应分子YAP/TAZ激活，这在实体癌中很常见。AMPK相关激酶NUAK2通过与LATS1相互作用被确立为Hippo信号的正向调控因子，但支配这种相互作用的确切机制仍不明确。我们的研究旨在鉴定NUAK2的新型近端相互作用蛋白，以发现控制Hippo通路输出的新调控复合物。
方法：我们利用BioID-质谱筛选鉴定NUAK2的近端相互作用蛋白。使用免疫共沉淀和GST下拉实验验证蛋白-蛋白相互作用。使用AlphaFold预测潜在的结构界面，并通过Ni-NTA亲和纯化验证复合物的物理组装。在2D癌细胞系和3D结直肠癌类器官中，通过Hippo靶基因的RT-qPCR、YAP/TAZ定位的免疫荧光显微镜和细胞生长实验评估DLG5缺失对Hippo信号的影响。
结果：我们的蛋白质组学筛选将支架蛋白DLG5鉴定为一个突出且新颖的NUAK2相互作用蛋白。我们随后验证DLG5支架化组装了一个三聚体复合物，在亲和纯化实验中与NUAK2和核心Hippo激酶LATS1共洗脱。在功能上，敲低DLG5破坏了Hippo信号，导致YAP/TAZ胞质定位，并在细胞系和CRC患者来源类器官中相应地降低了经典YAP/TAZ靶基因（如ANKRD1、CTGF）的转录。DLG5的缺失也抑制了癌细胞的生长，这与NUAK2抑制一致。
结论：我们的发现揭示了一个此前未知的调控轴，其中DLG5作为支架来组装DLG5-NUAK2-LATS1复合物。这种支架化使NUAK2能够与LATS1相互作用，从而调控Hippo信号。我们的工作确定了DLG5在Hippo通路中的新作用，未来的结构研究将进一步定义该复合物的分子构筑，以指导治疗性靶向。
查看英文原文 English abstract
Introduction : The Hippo signaling pathway is a critical regulator of cell growth and organ size, and its dysregulation, leading to activation of the transcriptional effectors YAP/TAZ, is common in solid cancers. The AMPK-related kinase NUAK2 is an established positive regulator of Hippo signaling through its interaction with LATS1, yet the precise mechanisms governing this interaction remain undefined. Our research aimed to identify novel proximal interactors of NUAK2 to uncover new regulatory complexes that control Hippo pathway output.
Methods : We utilized a BioID-mass spectrometry screen to identify NUAK2 proximal interactors. Validation of protein-protein interactions was performed using co-immunoprecipitation and GST pull-down assays. The potential structure interface was predicted using AlphaFold, and the physical assembly of the complex was validated via Ni-NTA affinity purification. The effect of DLG5 loss on Hippo signaling was assessed in both 2D cancer cell lines and 3D colorectal cancer organoids by RT-qPCR of Hippo target genes, immunofluorescence microscope for YAP/TAZ localization, and cell growth assays.
Results : Our proteomic screen identified the scaffolding protein DLG5 as a prominent and novel NUAK2 interactor. We subsequently validated that DLG5 scaffolds a trimeric complex, co-eluting with both NUAK2 and the core Hippo kinase LATS1 in affinity purification assays. Functionally, knockdown of DLG5 disrupted Hippo signaling, leading to YAP/TAZ cytoplasmic localization and a corresponding decrease in the transcription of canonical YAP/TAZ target genes (e.g., ANKRD1, CTGF ) in cell lines and CRC patient-derived organoids. The loss of DLG5 also suppressed cell growth in cancer cells, consistent with NUAK2 inhibition.
Conclusion : Our findings reveal a previously unknown regulatory axis where DLG5 acts as a scaffold to assemble the DLG5-NUAK2-LATS1 complex. This scaffolding allows NUAK2 to interact with LATS1, thereby regulating Hippo signaling. Our work identifies a novel role of DLG5 in the Hippo pathway, and future structural studies will further define the molecular architecture of this complex to guide therapeutic targeting.
利益披露 Disclosure
Y. Song, None..
A. Ogunjimi, None..
S. Song, None..
J. L. Wrana, None..
L. Attisano, None.