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AKT1, LKB1, and YAP1 Revealed as MYC Interactors with NanoLuc-Based Protein-Fragment Complementation Assay
Xiulei Mo1, Qi Qi1, Andrei A Ivanov1
1Department of Pharmacology and Emory Chemical Biology Discovery Center (X.M., Q.Q., A.A.I., Q.N., Y.L., J.H., R.G., S.B., M.A.J., Y.D., H.F.) and Department of Biomedical Informatics (L.A.D.C.), Emory University School of Medicine, Atlanta, Georgia; Departments of Hematology and Medical Oncology (F.R.K., H.F.) and Pathology and Laboratory Medicine (C.S.M.) and Winship Cancer Institute, Emory University, Atlanta, Georgia; State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China (Y.L.); and Department of Biomedical Engineering, Emory University School of Medicine/Georgia Institute of Technology, Atlanta, Georgia (L.A.D.C.).
Abstract:
The c-Myc (MYC) transcription factor is a major cancer driver and a well-validated therapeutic target. However, directly targeting MYC has been challenging. Thus, identifying proteins that interact with and regulate MYC may provide alternative strategies to inhibit its oncogenic activity. In this study, we report the development of a NanoLuc-based protein-fragment complementation assay (NanoPCA) and mapping of the MYC protein interaction hub in live mammalian cells. The NanoPCA system was configured to enable detection of protein-protein interactions (PPI) at the endogenous level, as shown with PRAS40 dimerization, and detection of weak interactions, such as PINCH1-NCK2. Importantly, NanoPCA allows the study of PPI dynamics with reversible interactions. To demonstrate its utility for large-scale PPI detection in mammalian intracellular environment, we have used NanoPCA to examine MYC interaction with 83 cancer-associated proteins in live cancer cell lines. Our new MYC PPI data confirmed known MYC-interacting proteins, such as MAX, GSK3A, and SMARCA4, and revealed a panel of novel MYC interaction partners, such as RAC-α serine/threonine-protein kinase (AKT)1, liver kinase B (LKB)1, and Yes-associated protein (YAP)1. The MYC interactions with AKT1, LKB1, and YAP1 were confirmed by coimmunoprecipitation of endogenous proteins. Importantly, AKT1, LKB1, and YAP1 were able to activate MYC in a transcriptional reporter assay. Thus, these vital growth control proteins may represent promising MYC regulators, suggesting new mechanisms that couple energetic and metabolic pathways and developmental signaling to MYC-regulated cellular programs.
Insights
Researchers developed a novel assay to map protein interactions with the cancer-driving transcription factor c-Myc (MYC). This identified new MYC regulators, including AKT1, LKB1, and YAP1, offering potential new therapeutic strategies against cancer.
Area of Science:
- Molecular Biology
- Cancer Research
- Cellular Signaling
Background:
- The c-Myc (MYC) transcription factor is a critical driver of cancer, presenting a significant therapeutic challenge.
- Directly targeting MYC is difficult, necessitating alternative strategies like identifying its regulatory protein interactions.
- Understanding MYC's interaction network is key to inhibiting its oncogenic functions.
Purpose of the Study:
- To develop and validate a novel assay for mapping protein-protein interactions (PPIs) of MYC in live cells.
- To identify novel MYC-interacting proteins and regulators within the intracellular environment.
- To explore new therapeutic strategies by understanding MYC's regulatory network.
Main Methods:
- Development of a NanoLuc-based protein-fragment complementation assay (NanoPCA) for detecting PPIs in live mammalian cells.
- Application of NanoPCA for large-scale mapping of MYC interactions with 83 cancer-associated proteins.
- Validation of novel MYC interactions using coimmunoprecipitation and functional assays.
Main Results:
- The NanoPCA system successfully detected endogenous and weak protein interactions, including PRAS40 dimerization and PINCH1-NCK2 interaction.
- Confirmed known MYC interactors (MAX, GSK3A, SMARCA4) and identified novel partners: AKT1, LKB1, and YAP1.
- Demonstrated that AKT1, LKB1, and YAP1 activate MYC, suggesting their role as key regulators.
Conclusions:
- The developed NanoPCA is a powerful tool for studying PPIs, including dynamic and weak interactions, in live cells.
- Novel MYC regulators AKT1, LKB1, and YAP1 were identified, highlighting their potential as therapeutic targets.
- These findings reveal new links between metabolic pathways, developmental signaling, and MYC-driven cancer programs.
