Rapid Detection of Dynamic PTEN Regulation in Living Cells Using Intramolecular BRET.

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.

A biosensor to monitor dynamic regulation and function of tumour suppressor PTEN in living cells.

Tumour suppressor PTEN is a phosphatase that negatively regulates the PI3K/AKT pathway. The ability to directly monitor PTEN conformation and function in a rapid, sensitive manner is a key step towards developing anti-cancer drugs aimed at enhancing or restoring PTEN-dependent pathways. Here we developed an intramolecular bioluminescence resonance energy transfer (BRET)-based biosensor, capable of detecting signal-dependent PTEN conformational changes in live cells. The biosensor retains intrinsic properties of PTEN, enabling structure-function and kinetic analyses. BRET shifts, indicating conformational change, were detected following mutations that disrupt intramolecular PTEN interactions, promoting plasma membrane targeting and also following physiological PTEN activation. Using the biosensor as a reporter, we uncovered PTEN activation by several G protein-coupled receptors, previously unknown as PTEN regulators. Trastuzumab, used to treat ERBB2-overexpressing breast cancers also elicited activation-associated PTEN conformational rearrangement. We propose the biosensor can be used to identify pathways regulating PTEN or molecules that enhance its anti-tumour activity.