The long-term goal of this proposal is to understand how specific protein:protein interactions regulate the localization of protein kinase C (PKC). This localization is of paramount importance in poising PKC near its co-factors, substrates, and regulators. PKC plays a pivotal role in transducing the myriad of hormonal, sensory, and neurotransmitter signals that promote phospholipid hydrolysis. The enzyme is regulated by two distinct, but equally critical, mechanisms: phosphorylation and lipid co-factors. Each regulates both the subcellular localization of the enzymes and the activity of the enzyme. Specifically, phosphorylation on two regions of the kinase core is required first to structure the active site for catalysis (transphosphorylation), and second to release PKC from cytoskeletal components into the cytosol (autophosphorylation). Generation of diacylglycerol recruits PKC to the membrane via the kinase's two membrane-targeting domains; this membrane interactions result in removal of the auto-inhibitor (pseudosubstrate) domain from the active site. Mounting evidence suggest that the subcellular localization of both inactive and activated PKC may be further fine-tuned by interaction with specific anchoring of targeting proteins. The goal of the proposed research is to understand how non-phosphorylated PKC is tethered to the cytoskeleton and how phosphorylation releases this anchorage, how mature PKC interacts with a known anchoring protein, gravin, and lastly, how disruption of these protein:protein interactions affects signal transduction.
Three Specific Aims are: 1. Mechanism of tethering on-phosphorylated PKC to the cytoskeleton. The goal addressed the mechanism of tethering non-phosphorylated PKC to the cytoskeleton. Specifically, experiments are proposed to identify the cytoskeletal binding protein for PKC, to understand the molecular basis for this interaction, and to elucidate how phosphorylation disrupts this interaction. 2. Mechanism of interaction of phosphorylated PKC with anchoring protein gravin. The goal of this aim is to understand the mechanism by which mature PKC interacts with a known targeting protein, gravin. 3. Role of anchoring in PKC function. The goal of this aim is to determine how disruption of PKC anchoring affects cell signaling using lamin B phosphorylation as a read-out for PKC function.
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