Defining a DLG5-NUAK2-LATS1 complex that regulates Hippo signaling output

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.