Stimulation of
tyrosine kinase receptors initiates a signaling cascade that activates PI3K. Activated PI3K uses PIP2 to generate PIP3, which recruit Akt to the plasma membrane through its
pleckstrin homology (PH) domain, permitting its activation by PDKs. Activated Akt controls important
biological functions, including cell metabolism, proliferation and survival. The PI3K pathway is therefore an attractive target for
drug discovery. However, current assays for measurement of PIP3 production are technically demanding and not amenable to high-throughput screening. We have established a MCF-7-derived
breast cancer cell line, that stably co-expresses the PH domain of Akt fused to
Renilla luciferase and YFP fused to a membrane localization signal. This BRET biosensor pair permits to monitor, in real time, in living cells, PIP3 production at the plasma membrane upon stimulation by different
ligands, including
insulin, the
insulin analogue
glargine, IGF1, IGF2 and
EGF. Moreover, several known inhibitors that target different steps of the PI3K/Akt pathway caused inhibition of
ligand-induced BRET.
Cetuximab, a humanized anti-
EGF receptor monoclonal antibody used for the treatment of
cancer, completely inhibited
EGF-induced BRET, and the
tyrosine kinase inhibitor tyrphostine
AG1024 inhibited
insulin effect on PIP3 production. Moreover, the effects of
insulin and IGF1 were inhibited by molecules that inhibit PI3K catalytic activity or the interaction between PIP3 and the PH domain of Akt. Finally, we showed that human serum induced a dose-dependent increase in BRET signal, suggesting that this stable clone may be used as a prognostic tool to evaluate the PI3K stimulatory activity present in serum of human patients. We have thus established a cell line, suitable for the screening and/or the study of molecules with stimulatory or inhibitory activities on the PI3K/Akt pathway that will constitute a new tool for translational research in diabetes and
cancer.