The PKC family of Ser/Thr kinases transduces the abundance of extracellular signals that promote phospholipid hydrolysis. The long-term goal of this proposal is to understand how specific protein-protein interactions regulate signaling by protein kinase C. This proposal focuses on investigating the function of novel interactions between the C-terminal PDZ ligand of the conventional PKC isozyme, PKC alpha, and the membrane-associated guanylate kinase (MAGUK) family of PDZ domain-containing scaffolds. MAGUK proteins are central to coordinating signaling complexes at cell-cell junctions. Two MAGUK protein scaffolds, PSD-95 and SAP97, coordinate signaling at excitatory synapses, a specialized cell junction in neurons involved in memory and learning. We have identified these two scaffolds as PKC alpha interacting partners. PKC alpha has been shown to regulate synaptic plasticity by phosphorylating a number of substrates, including glutamate receptors, which are themselves scaffolded to MAGUK proteins via PDZ interactions. Phosphorylation of glutamate receptors by PKC increases the number and activity of the receptors at the synapse. Our preliminary data has verified the PDZ-dependent interaction between the MAGUK proteins and PKC alpha in an overexpression system. The next step will be to demonstrate that scaffolding to MAGUKs regulates the phosphorylation of PKC alpha substrates; this will be carried out using previously described phospho-specific antibodies. We also plan to use our expertise in imaging localized signal transduction to target a genetically encoded sensor for PKC activity to MAGUK protein scaffolds to read out PKC activity at these assemblies in live cells. Finally, we will develop a peptide that specifically disrupts the interaction between PKC alpha and the MAGUK proteins. The goal of this is to tease apart the synaptic functions of PKC alpha mediated by MAGUK scaffolding versus PICK1, another PDZ domain- containing protein at synapses that constitutes PKC alpha's only other known PDZ interaction. Signaling by the protein kinase C family underlies a multitude of physiologically relevant processes, including cell proliferation, immune system function, and memory. Consequently, mis-regulation of PKC signaling underlies many disease processes, notably cancer. Understanding the specifics of PKC regulation by protein:protein interactions will undoubtedly result in better, more specific therapeutic targets for disease pathways involving PKC.
