Protein kinase C (PKC) has been implicated as a component of the Nerve growth factors (NGF) signalling pathway in PC12 cells; although, disagreement regarding the role that PKC plays in this process has persisted in the literature. We provide novel information demonstrating that activation of atypical zeta-PKC relates to the signalling paradox surrounding NGF and PKC isoforms. In addition, we also document that zeta- PKC is activated by NGF in a trk-dependent manner and modulated in a positive fashion by increases in cAMP. We hypothesize that zeta-PKC lies in parallel with raf kinases and is a major constituent of a newly described signalling pathway serving as a MAP kinase kinase kinase (MEK kinase). The goal of this project is to precisely define the role which zeta-PKC plays in mediating signal-coupling and neurite outgrowth. In order to fulfill this goal, two specific aims will be undertaken. We are proposing to; 10 establish the means by which activation of zeta-PKC modulates an existing or newly defined pathway by studying its interaction with proteins which comprise athe classical NGF signalling cascade involving ras / raf yieldsMAP kinase and 2) overexpress zeta-PKC to upregulate its signal and study both biochemical and morphological aspects of overexpression. The long-term goal of these studies is to establish the mechanism whereby zeta-PKC regulates NGF dependent and independent signalling involving neuronal differentiation. To accomplish these aims we will employ PC12 cells deficient in zeta-PKC or those overexpressing this isoform. The regulation exerted by zeta-PKC will be studied using a range of techniques: in situ activity assays, immunoprecipitation of individual signaling components, reconstitution, protein purification, peptide mapping and phosphoamino acid analysis. The outcome of these studies stand to change our conceptual framework of NGF-signal coupling and neurite outgrowth. Moreover, the findings generated by this study will have a direct impact upon Alzheimer's research, since PC12 cells have been used as a model system to elucidate the role of amyloid precursor protein (APP). Previous work in PC12 cells has demonstrated that APP is capable of enhancing neurite outgrowth and acts as a mediator of NGF's effects. Hence, elucidating the signalling pathway(s) leading to neurite extension will bear directly into the etiology and treatment of Alzheimer's, as well as, other human disease in which alterations in PKC and NGF are hypothesized to play a role.