Where does all the PIP2 come from?

Abstract

Despite its very low concentration in the plasma membrane, PIP(2) is the precursor for the important second messenger InsP(3) and, independently, is a key modulator of membrane signalling molecules such as ion channels. However, it has been difficult to determine the spatial and temporal characteristics of PIP(2) and InsP(3) during a cell signalling event. Our laboratory used bradykinin stimulation of N1E-115 neuroblastoma cells to infer the InsP(3) dynamics from calcium imaging studies, biochemical analysis and InsP(3) uncaging. We have used computational modelling with Virtual Cell to help analyse and interpret experimental data on the details of the calcium release process as well as to build a comprehensive image-based model of agonist-induced calcium release in a neuronal cell. These data provided a constraint for the further investigation of how low levels of cellular PIP(2) could provide sufficient InsP(3) for calcium release. Using biochemical assays, quantitative imaging of GFP-based probe translocation and computational analysis, it was shown that PIP(2) synthesis is stimulated concomitant with its hydrolysis. This mechanism should be important not just for consideration of PIP(2) as a precursor of InsP(3), but for any pathway that can be directly or indirectly modulated by PIP(2).