Differential activation of the inositol 5-phosphatase SHIP2 by EGF and insulin signaling pathways.

The importance of phosphatidylinositol 3,4,5- trisphosphate (PIP(3)) in cell signaling has been well established. Despite phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] emerging as an actor independent of PIP(3), its exact signaling role remains poorly understood, and the precise dynamics of PI(3,4)P(2) and PIP(3) upon receptor tyrosine kinase (RTK) stimulation are still inadequately investigated. In this study, we employed bioluminescence resonance energy transfer (BRET) sensors to monitor plasma membrane phosphoinositide (PIP) dynamics in HEK293-derived and HeLa cells following stimulation with epidermal growth factor (EGF) and insulin. Our findings reveal significant differences in PIP regulation: The increase in PI(3,4)P(2) compared to PIP(3) was larger with EGF stimulation relative to insulin. Using siRNA-mediated knockdown, we identified SH2-domain containing inositol polyphosphate 5-phosphatase 2 (SHIP2) as the key enzyme responsible for PI(3,4)P(2) production in the EGF pathway, which was further supported by a bioinformatics analysis. Moreover, we detected increased phosphorylation at two tyrosine sites in SHIP2 upon EGF stimulation, which was shown to be dependent on PI3K activation and PLC-induced calcium signal. These findings help refine our understanding of receptor-specific phosphoinositide dynamics and the enzymatic machinery involved, as well as their potential influence on downstream cellular responses.