Dual compartment utility of BRET-based biosensors for PPP2R5A/B56α, a cancer-associated B regulatory subunit of PP2A

Protein phosphatase 2A (PP2A), a pivotal serine/threonine phosphatase, plays a crucial role in cellular regulation and tumor suppression. Dysregulation of PP2A complex, particularly the Aα subunit and B56 family, is linked to malignancies through altered substrate interactions, exemplified by c-MYC dynamics. Given the challenges in identifying PP2A substrates—owing to the enzyme’s expansive substrate range, transient interaction profiles, and complex regulatory mechanisms—we employed bioluminescence resonance energy transfer (BRET) sensors. These advanced molecular tools facilitate the real-time detection of protein-protein interactions within live cells. This investigation details the creation and application of a novel PPP2R5A (B56α) BRET sensor tailored for cytosolic and nuclear environments, effectively distinguishing specific PP2A interactions. The nuclear sensor, enhanced with a nuclear localization signal, enabled probing of targets like c-MYC. The dual compartmental utility of these sensors underscores their significant potential in elucidating PP2A’s regulatory roles and their implications in oncogenesis. Our study highlights the efficacy of BRET sensors in formulating precision therapeutic strategies. This advancement provides a robust framework for deeper investigations into the multifaceted roles of PP2A in both normal physiological and pathological contexts, paving the way for future explorations into its intricate molecular interactions. HEK293T cells were transfected with plasmids encoding NanoLuc- or YFP-tagged proteins, with or without a nuclear localization signal (NLS), to evaluate protein–protein interactions in the cytoplasm and nucleus using BRET assays. To confirm correct subcellular localization, we performed western blotting on nuclear and cytoplasmic fractions, as well as immunofluorescence imaging using a confocal microscope. BRET signals were measured following luminescent substrate addition. The BRET results were further validated by immunoprecipitation assays.

蛋白磷酸酶2A（Protein phosphatase 2A, PP2A）作为一类关键的丝氨酸/苏氨酸磷酸酶，在细胞调控与肿瘤抑制过程中发挥着至关重要的作用。PP2A复合物的失调——尤其是其Aα亚基与B56家族——可通过改变底物相互作用模式诱发恶性肿瘤，c-MYC动力学异常便是典型例证。鉴于PP2A底物的鉴定存在诸多挑战：该酶的底物谱范围广泛、相互作用具有瞬时性且调控机制复杂，本研究采用了生物发光共振能量转移（Bioluminescence resonance energy transfer, BRET）传感器。这类先进的分子工具可实现活细胞内蛋白质-蛋白质相互作用的实时检测。本研究详细介绍了一款针对胞质与核环境定制开发的新型PPP2R5A（B56α）BRET传感器，其可有效区分特异性PP2A相互作用。其中添加了核定位信号的核定位传感器，可用于探究c-MYC等靶标蛋白。这类具备双亚细胞分布特性的传感器，为阐明PP2A的调控功能及其在肿瘤发生中的作用提供了巨大潜力。本研究证实了BRET传感器在制定精准治疗策略方面的有效性。该研究进展为深入探究PP2A在生理正常与病理状态下的多重功能提供了可靠框架，也为未来探索其复杂的分子相互作用铺平了道路。本研究将编码NanoLuc或YFP标签蛋白（带有或不带有核定位信号（NLS））的质粒转染至HEK293T细胞中，通过BRET实验评估胞质与细胞核内的蛋白质-蛋白质相互作用。为验证正确的亚细胞定位，我们对核组分与胞质组分进行了蛋白质免疫印迹（Western blotting）分析，并通过共聚焦显微镜开展免疫荧光成像。在添加发光底物后检测BRET信号。此外，通过免疫沉淀实验进一步验证了BRET实验结果。