| dc.description.abstract | Protein Kinase C (PKC) family of isoenzymes are critical players in signal transduction at the membrane surface. Its aberrant activity has been associated with Alzheimer’s disease, cancer, and mood disorders. Significant efforts have been made to understand how PKC is regulated in order to design drugs to modulate activity. This has proved challenging due to the dynamic nature of PKCs.
The key event in PKC activation is the translocation of its regulatory region to anionic membranes in response to second messengers. This region in conventional isozymes contains three peripheral membrane binding modules: twin C1 domains that penetrate the membrane in response to diacylglycerol and a C2 domain that binds to anionic lipids upon association with up to three Ca2+ions. The objective of this dissertation is to provide mechanistic insight into PKC activation by probing the regulatory region’s structure, function, and interactions with toxic substances. Paramagnetic solution NMR revealed that the C1B-C2 region from PKC primarily samples open conformations, with both membrane binding sites exposed. In several of these conformations, the membrane binding loops of C1B and C2 are in relative orientations that are compatible with simultaneous membrane insertion. This suggested that C1 and C2 domains coordinate membrane binding. Consistent with this, functional studies showed that C1 ligands lowered the amount of Ca2+ needed to traffic C2 to membrane mimics.
Studies with membrane mimics revealed that the regulatory region responded differently to C1 ligands, diacylglycerol and tumor-promoting phorbol ester, PDBu. Preliminary data indicated that diacylglycerol drives the C1 domain deeper into the bilayer. Furthermore, the presence of the low abundance signaling lipid, phosphatidylinositol 4,5-bisphosphate, was required for bivalent binding of C1 and C2 in the presence of diacylglycerol but not PDBu. Finally, previous studies have shown that PKCs are targeted by xenobiotic metal ions, Pb^2+ and Cd^2+. This work shows that both metals drive self-assembly of the C1BC2 regulatory region into large oligomers, enhancing membrane binding functionality. A new mode of Pb2+ and Cd2+ binding is identified. Both bind opportunistically to the linker region and C-terminal helix. Collectively this work adds to the understanding of PKC activation and its interactions with toxic substances. | en |
