Unbiased Functional Proteomics Strategy for Protein Kinase Inhibitor Validation and Identification of bona fide Protein Kinase Substrates: Application to Identification of EEF1D as a Substrate for CK2

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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