Imprimer

A biosensor to probe Tumour Suppressor PTEN function in live cells

PTEN Tumour Suppressor is a non-redundant phosphatase that acts as a brake on the oncogenic pro-survival PI3K/AKT pathway. Dysregulation of PTEN protein contributes to the development of a wide range of cancers.

NatComm

A biosensor to probe Tumour Suppressor PTEN function in live cells

 

            PTEN Tumour Suppressor is a non-redundant phosphatase that acts as a brake on the oncogenic pro-survival PI3K/AKT pathway. Dysregulation of PTEN protein contributes to the development of a wide range of cancers. PTEN activity in human cancer can be downregulated via posttranslational mechanisms in the absence of any PTEN gene alteration, thus providing potential therapeutic avenues to explore and exploit. The capacity to directly probe PTEN conformational rearrangement and function in a rapid, sensitive manner is therefore a key step towards developing anti-cancer drugs aimed at enhancing or restoring PTEN-dependent pathways. 

            We developed a Bioluminescence Resonance Energy Transfer (BRET)-based biosensor capable of reporting conformational rearrangement of PTEN in live cells. For this, PTEN was "sandwiched" between the energy donor, Renilla luciferase and the energy acceptor Yellow Fluorescent Protein. The biosensor retains intrinsic PTEN properties, enabling structure-function and kinetic analyses. Changes in BRET signals, indicating conformational change, were detected following targeted mutation of phosphorylation sites affecting key intramolecular interactions, as well as cancer-associated mutations ablating major mono-ubiquitination sites.  Using the biosensor as reporter we were able to detect signal-induced changes in PTEN conformation associated with its activation.  These include i) novel G protein-coupled receptor-mediated regulation, ii) pharmacological modulation of pathways that impact PTEN function, and 3) protein partners that play a role in controlling PTEN plasticity. Finally, Trastuzumab used to treat ERBB2-overexpressing breast cancers, also elicited activation-associated PTEN conformational change in various ERBB2-overexpressing cell lines. We propose the biosensor can be used as a platform to identify pathways regulating PTEN or molecules that enhance its anti-tumour activity.

This work was supported by the Fondation ARC pour la Recherche sur le Cancer, INSERM, CNRS and PhD fellowships of ARC and LABEX "WhoamI?"

 

Source


Lima-Fernandes E, Misticone S, Boularan C, Paradis JS, Enslen H, Roux PP, Bouvier M, Baillie GS, Marullo S & Scott MGH.
A Biosensor to monitor Dynamic Regulation and Function of Tumour Suppressor PTEN in Living Cells.
Nature Communications 2014 5 4431

 

Team: Stefano Marullo "Receptor Signalling & Molecular Scaffolds"

Contact:  Mark Scott, CNRS Research Associate, email: