Second messenger regulated membrane targeting: Membrane docking mechanisms of C2 and PH domains

C2 and pleckstrin homology (PH) domains serve to target eukaryotic proteins to distinct cellular membranes in response to transient calcium and phosphatidylinositol-phosphate second messenger signals, respectively. The protein-lipid interactions mediated by these domains are fundamental to the spatial and temporal regulation of signaling pathways at membrane surfaces during processes such as chemotaxis. For example, in polarized cells the kinases PKCalpha and AKT1 are recruited to the leading edge plasma membrane via interactions between their respective C2 and PH domains and negatively charged phospholipid targets. As a result, the activity of these kinases is spatially restricted to facilitate actin polymerization and membrane remodeling at the leading edge. Despite observations of dynamic membrane targeting of signaling proteins, descriptions of the underlying protein-lipid interactions that mediate this process are not well characterized. This research focuses on uncovering the molecular mechanisms of phospholipid binding that govern second messenger regulated, plasma membrane docking of the PKCalpha C2 and AKT1 PH domains. Electron paramagnetic resonance (EPR) spectroscopy was utilized to model the structure of the PKCalpha C2 domain docked to a plasma membrane surface in its high affinity state and provide the first detailed analysis of a lipid induced change in docking geometry important for Ca2+-activated, plasma membrane targeting specificity. In vitro and live cell fluorescence spectroscopy techniques were employed to elucidate the molecular mechanism of a cancerous mutation found in the AKT1 PH domain that alters lipid-binding specificity. Characterization of this charge reversal mutant revealed new insights into phosphatidylinositol-(3,4,5)-trisphosphate binding by the AKT1 PH domain as well as other PH domains. These studies provide detailed molecular descriptions of the protein-phospholipid interactions that govern membrane docking of the PKCalpha C2 and AKT1 PH domains and result in specific targeting to the cell plasma membrane. Not only are these findings critical to the function of the PKCalpha and AKT1 kinases, but also provide a framework from which to build an understanding of how related C2 and PH domains have optimized membrane-targeting mechanisms. Furthermore, the membrane docking mechanism of the PKCalpha C2 domain is applied to the development of a FRET-based sensor to measure calcium signals in polarized cells.