Although extracellular signal-regulated kinase (ERK) activation was originally thought to operate as a simple ON-OFF switch governing proliferation, it is now clear that duration of kinase activation can encode specific cellular responses. This phenomonon is best shown using the rat pheochromocytoma cell line, PC12 cells. In this model, nerve growth factor (NGF) produces sustained ERK activation resulting in differentiation. One mechanism underlying NGF's action is regulation of nuclear effectors, such as c-fos. However, a role for c-fos as a functional effector of NGF's sustained ERK activation has not been established. This proposal will examine the requirement for sustained activation of the ERK cascade in NGF-mediated c-fos regulation (Aim #1). In addition, the role of the small G protein Rap1 in NGF's actions will be investigated (Aim #2). We will be measuring activator protein-1-dependent gene expression and NGF-mediated c-fos protein stabilization as a readout of c-fos function. In addition, we will test the requirement of specific ERK phosphorylation sites in c-fos for NGF-dependent stabilization and transactivation in PC12 cells. This work will help us to further understand the regulation of neuronal differentiation.