Abstract

Phosphoinositide 3-kinase (PI3K) is a central component of numerous cellular signaling pathways, regulating critical processes such as cell growth, proliferation, and survival. Dysregulation of PI3K activity is implicated in various human diseases, making its real-time monitoring essential for both basic research and drug discovery. This study addresses the challenge of observing dynamic PI3K activation in living cells by developing a stable monoclonal line of HEK-293 cells expressing a genetically encoded sensor. The sensor, likely based on the Pleckstrin Homology (PH) domain of Akt, specifically detects the PI3K product, phosphatidylinositol (3,4,5)-trisphosphate (PIP3), allowing for fluorescent readout of enzyme activity. Upon stimulation with specific agonists, the engineered HEK-293 cells demonstrated robust and quantifiable activation of PI3K. Furthermore, the stable cell line, which in some related studies also incorporates a calcium sensor, enables the simultaneous analysis of PI3K activity and other key signaling events, providing a more comprehensive view of the cellular response. This novel, genetically encoded biosensor system provides a powerful, non-invasive tool for high-throughput screening of PI3K modulators and offers new insights into the spatio-temporal dynamics and cross-talk of PI3K signaling in a physiologically relevant cellular context.