Signaling cascades direct information transmission and, in turn, function in processes as diverse as memory and muscle movement. These cascades are mediated by a network of highly specific and tightly regulated protein:protein interactions, including those made by serine/threonine kinases and serine/threonine phosphatases. Our long-term goal is to achieve an in-depth understanding of signaling networks with a special focus on serine/threonine phosphatase signaling, by using biochemical, biophysical, structural biology, especially NMR spectroscopy and in vivo technologies. In the proposed studies, we will focus on the role of the serine/threonine phosphatase Protein Phosphatase 1 (PP1) in dendritic spine signaling, as it is one of the most important protein phosphatases in brain tissue. Two of the major interaction partners of PP1 in neurons are the large, multi-domain scaffolding proteins spinophilin and neurabin. These proteins target PP1 to its cellular point of action, the post synaptic density of dendritic spines. This targeting of PP1 by spinophilin and neurabin is responsible for the PP1-mediated regulation of glutamatergic AMPA/NMDA receptor activity and trafficking. To understand the spinophilin:PP1 and neurabin:PP1 signaling networks in molecular detail, we will use NMR spectroscopy to elucidate the 3-dimensional structures and scaffolding properties of the spinophilin and neurabin protein interaction domains, both as isolated domains and in complex with their protein binding partners. Based on the results obtained from the NMR studies, we will investigate the in vivo functional significance of these interactions in cells and neurons. These in vivo studies will be done in close collaboration with the Nairn laboratory at Yale University. Furthermore, neurabin and spinophilin are capable of inducing cytoskeletal remodeling by means of modulating f-actin organization and by activity-induced profilin targeting. Activity-induced profilin targeting plays a major role in the stabilization of the dendritic spine morphology and thus long-term memory. We will first generate biochemical and structural in vitro data of these biologically critical events, and subsequently test our models using in vivo functional assays to provide a detailed understanding of critical parameters in dendritic spine signaling and morphology. These structural and functional studies will provide a detailed understanding of the roles of spinophilin and neurabin in brain and will, as a long term goal, enable us to selectively modulate these particular signaling cascades for medical benefit. ? ? ?
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