PhD Scientific Days 2024

Molecular Medicine II.

Differential Activation of SHIP2 by EGF and Insulin Signaling Pathways

Text of the abstract

The importance of phosphatidylinositol 3,4,5- trisphosphate (PIP3) in biological processes has been well established. Among the known phosphoinositides (PIs), the precise role of phosphatidylinositol 3,4- bisphosphate (PtdIns(3,4)P2) and its relationship to PIP3 in signal transduction has remained poorly understood. In this study, we investigated the molecular mechanism responsible for the production of PtdIns(3,4)P2 upon EGF and insulin stimulation in HEK cells. We largely rely on our recently developed bioluminescent resonance energy transfer (BRET) sensors for detection and quantification of various PIs. Here we demonstrate a striking variation in the resultant levels of PIP3 and PtdIns(3,4)P2 with EGF compared to insulin stimulation. We demonstrate that the increase of the PtdIns(3,4)P2 compared to PIP3 is much greater in the setting of EGF. Using siRNA- mediated knockdown and immunoblotting we show the importance and increased activity of the SHIP2 enzyme, a 5- phosphatase enzyme which converts PIP3 to PtdIns(3,4)P2, in the EGF signal transduction pathway. Furthermore, using pretreatment with the PI3K inhibitor wortmannin and the calcium chelator BAPTA-AM we demonstrate that the EFF-induced activation of SHIP2 enzymes is dependent on both the PI3Ks activity and the calcium signaling it initiates. Our findings help refine our understanding of the distinct consequences elicited by EGF and insulin signaling.