AKT1, LKB1, and YAP1 Revealed as MYC Interactors with NanoLuc-Based Protein-Fragment Complementation Assay
AKT1, LKB1, and YAP1 Revealed as MYC Interactors with NanoLuc-Based Protein-Fragment Complementation Assay
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DOI：
10.1124/mol.116.107623
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发表时间：
2017-04-01
影响因子：
3.6
通讯作者：
Fu, Haian
中科院分区：
文献类型：
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作者：
Mo, Xiulei;Qi, Qi;Fu, Haian
c-Myc (MYC) 转录因子是主要的癌症驱动因素，也是经过充分验证的治疗靶点。然而，直接针对 MYC 一直具有挑战性。因此，鉴定与 MYC 相互作用并调节 MYC 的蛋白质可能提供抑制其致癌活性的替代策略。在这项研究中，我们报告了基于 NanoLuc 的蛋白质片段互补测定 (NanoPCA) 的开发以及活哺乳动物细胞中 MYC 蛋白质相互作用中心的图谱。 NanoPCA 系统配置为能够在内源水平检测蛋白质-蛋白质相互作用 (PPI)，如 PRAS40 二聚化所示，并检测弱相互作用，例如 PINCH1-NCK2。重要的是，NanoPCA 允许研究具有可逆相互作用的 PPI 动力学。为了证明其在哺乳动物细胞内环境中大规模 PPI 检测的实用性，我们使用 NanoPCA 来检查 MYC 与活癌细胞系中 83 种癌症相关蛋白的相互作用。我们的新 MYC PPI 数据证实了已知的 MYC 相互作用蛋白，如 MAX、GSK3A 和 SMARCA4，并揭示了一组新型 MYC 相互作用伙伴，如 RAC-α 丝氨酸/苏氨酸蛋白激酶 (AKT) 1、肝激酶 B (LKB) 1 和 Yes 相关蛋白 (YAP) 1。MYC 与 AKT1、LKB1 和 YAP1 的相互作用得到了证实内源蛋白的免疫共沉淀。重要的是，AKT1、LKB1 和 YAP1 能够在转录报告基因检测中激活 MYC。因此，这些重要的生长控制蛋白可能代表有前途的 MYC 调节剂，表明将能量和代谢途径以及发育信号传导与 MYC 调节的细胞程序相结合的新机制。
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-alpha 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.