Misticone Stanislas, Lima-Fernandes Evelyne, Scott Mark G H
Inserm, U1016, Institut Cochin, 27, Rue du Faubourg Saint Jacques, 75014, Paris, France.
CNRS, UMR8104, Paris, France.
Methods Mol Biol. 2016;1388:95-110. doi: 10.1007/978-1-4939-3299-3_8.
Tumor suppressor PTEN phosphatase acts to inhibit the PI3K/AKT pathway and thus regulates cell proliferation, survival, and migration. Dysregulation of PTEN function is observed in a wide range of cancers. In addition to alterations of the PTEN gene, repression of PTEN function can also occur at the protein level through changes in PTEN conformation, localization, activity, and stability. The ability to follow switches in PTEN conformation in live cells provides a rapid approach to study changes in PTEN function and may provide a basis to screen pharmacological agents aimed at enhancing or reestablishing PTEN-dependent signaling pathways that have gone awry in cancer. Here, we describe methods to use an intramolecular bioluminescent resonance energy transfer (BRET)-based biosensor that reports dynamic signal-dependent changes in PTEN conformational rearrangement and function.
肿瘤抑制因子PTEN磷酸酶可抑制PI3K/AKT信号通路，从而调节细胞增殖、存活和迁移。在多种癌症中均观察到PTEN功能失调。除了PTEN基因的改变外，PTEN功能的抑制也可能在蛋白质水平通过PTEN构象、定位、活性和稳定性的变化而发生。在活细胞中追踪PTEN构象变化的能力为研究PTEN功能变化提供了一种快速方法，也可能为筛选旨在增强或重建在癌症中出现异常的PTEN依赖性信号通路的药物提供依据。在此，我们描述了使用基于分子内生物发光共振能量转移（BRET）的生物传感器的方法，该传感器可报告PTEN构象重排和功能中动态的信号依赖性变化。
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