PKC gamma is one of the four members of the conventional PKC family, which are regulated by calcium and diacylglycerol. Unlike any other members of the family, PKC gamma has an unique expression pattern, being expressed predominantly in brain and spinal cord. Recently PKC gamma was also found in heart. The experiments proposed here are designed to answer two main questions. The first is what allows PKC gamma to respond to some signals but not others by phosphorylating unique substrates in response to each signal. Our working model is that PKC gamma is spatially confined in both its' states, active and inactive. Spatial localization is achieved by binding to particular proteins and the affinity of the binding is regulated by upstream signals. To test this hypothesis I will first identify binding proteins/substrates for the active and inactive PKC gamma by using the yeast two hybrid approach. I will then test the interaction in the native tissue and determine the factors that modulate the interaction. The second question is what are the physiological signals that activate PKC gamma in excitable cells. Is it activated by calcium influx through ion channels during elevated electrical activity of the neurons or is it exclusively regulated through metahotropic receptors? To answer these questions I will first design novel fluorescent indicators which will allow me to detect the activation of PKC gamma in a single cell assay. Using these indicators I will determine the spatial and temporal aspects of PKC gamma activation in excitable cells in response to a variety of stimuli.
