Site-specific phosphorylation of cytoskeletal proteins is of fundamental importance in normal neuronal function, and likely contributes to pathological dysfunction in certain neurodegenerative disorders, especially Alzheimer's disease. Tau and microtubule-associated protein 2 (MAP-2) are two critically important phosphorylated cytoskeletal proteins necessary for the maintenance of neuronal structure and function. The overall goals of this proposal are to use in vitro and in situ approaches to examine the phosphorylation of tau and MAP-2 to test the hypothesis that site specific phosphorylation events modulate the function and metabolism of these proteins. The specific goals of this project are to test the following hypotheses: (1) Site-specific phosphorylation of tau and MAP-2 by particular proline- directed protein kinases alters their susceptibility to degradation by the calcium-activated protease, calpain, (2) Stimulation of cAMP and Ca++- mediated signaling pathways in intact cells leads to activation of cAMP- dependent protein kinase and Ca++/calmodulin-dependent protein kinase II, respectively, and site-specific phosphorylation of tau and MAP-2, (3) specific growth factors (e.g., nerve growth factor) activate signaling pathways which results in the phosphorylation of tau and MAP-2, (4) site- specific phosphorylation of tau and MAP-2 in intact cells modulates their function, metabolism and localization, and (5) treatment of intact cells with certain growth factors modulates the synthesis and metabolism of tau and MAP-2, and this results in functional changes. This proposal represents a continuation of our highly successful previous studies examining the crucial role of site-specific on modulating the metabolism and function of tau and MAP-2. The goals of this proposal will be accomplished by using a unique combination of in situ and in vitro methods applied in this laboratory. One approach used in this study is to stimulate certain signaling pathways in situ to activate specific kinases and the phosphorylation of tau and MAP-2. In preliminary studies, activation of the cAMP-mediated signaling pathways and cAMP-dependent protein kinase resulted in increased phosphate incorporation into tau in rat brain slices. Additionally, treatment of P19 cells differentiated into a neuronal phenotype with nerve growth factor (NGF) resulted in increased phosphorylation of tau, apparently by proline- directed protein kinases. These and other preliminary findings clearly demonstrate that the studies outlined in this proposal will yield important new information on how the phosphorylation of tau and MAP-2 is modulated in situ, and the metabolic and functional consequences of site- specific phosphorylation of these proteins.