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

The importance of phosphatidylinositol 3,4,5- trisphosphate (PIP₃) in cell signaling has been well established. Despite phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂] emerging as an actor independent of PIP₃, its exact signaling role remains poorly understood, and the precise dynamics of PI(3,4)P₂ and PIP₃ 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)P₂ compared to PIP₃ 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)P₂ 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.