Reconstitution of Ras-PI3Kγ membrane communication and feedback using light-induced signaling inputs

Biochemical reactions involving phosphatidylinositol phosphate (PIP) lipids and small GTPases serve essential roles in signal transduction immediately downstream of cell surface receptor activation. Interconnected positive and negative feedback loops link these distinct classes of reactions and allow for the rapid establishment of the steep and precisely oriented intracellular activity gradients that are hallmarks of cell polarity. Although genetic approaches have identified numerous molecules that potentially regulate feedback between GTPases and PIP lipids in cells, it is unclear how different types of feedback modulate the spatiotemporal dynamics of membrane signaling reactions. Here, we reconstitute communication and feedback between Ras GTPase and phosphatidylinositol 3-kinase gamma (PI3K{gamma})-mediated generation of PIP3 on supported membranes. We employ light-induced membrane recruitment of a guanine nucleotide exchange factor (GEF) to rapidly and reversibly shift steady-state conditions and monitor corresponding Ras activation and PIP3 production in time and space. Alone, the Ras-PI3K{gamma} module exhibits weak and transient activation due to global inhibitors. However, the introduction of GEF-mediated positive feedback drives local amplification of Ras activation and PIP3 generation, resulting in a wave of activity that propagates across the membrane surface. Spatial coupling between activated Ras and PIP3 molecules depends on their respective diffusion coefficients and the species involved in the feedback circuit. This work identifies activation thresholds, membrane diffusion, and feedback architecture as key factors that determine how small GTPases and PIP lipid modifying enzymes amplify membrane signaling reactions in the presence of global inhibitors.