Elevated intracellular calcium is one of the most important signaling molecules in cells, especially in neural tissues. One of the important actions of calcium is to regulate the transcription of selected genes, thereby altering the phenotype of the cell. Studies from a number of laboratories, including our own, have demonstrated that a family of calmodulin-dependent protein kinases mediate many of these Ca2+- dependent transcriptional events. In particular, we have focused on CaM-kinase IV which can phosphorylate several transactivating proteins such as CREB and SRF. In the past two years we have partially characterized a CaM-kinase cascade consisting of a CaM- kinase kinase which can phosphorylate and activate CaM-kinase IV and CaM-kinase I. In the current grant application we will further characterize the involvement of this CaM-kinase cascade by: 1) identifying additional upstream components of the cascade (e.g. CaM- kinase kinase kinase) or anchoring proteins for CaM-kinase kinase. 2) identifying additional transactivating proteins for CaM-kinase IV by examining transcriptional regulation of the NGFI-B gene by the CaM- kinase cascade. 3) determining the sites and physiological relevance of cross-talk between the CaM-kinase cascade and the MAP-kinase cascade. These studies will be performed in a variety of cultured cells, especially PC12 cells, and will utilize biochemical and molecular biological techniques. The results of this study have implications for modulating cellular apoptosis and synaptic plasticity due to Ca2+- dependent gene transcription.