This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Neuronal growth cone guidance is driven by attractive or repulsive guidance cues that spatial coordinate actin cytoskeletal dynamics. Importantly, the balance between attraction and repulsion is modulated by cAMP and the cAMP-dependent protein kinase (PKA). Previously, we have shown that a subset of PKA regulatory subunits, as well as PKA activity, and the phosphorylation of a number of cytoskeletal targets are all significantly enriched in protruding pseudopodia and lamellipodia in fibroblasts. Moreover, inhibition of either PKA activity or PKA interaction with AKAPs (A-kinase anchoring proteins) prevents pseudopod formation and inhibits chemotaxis. This suggests that cytoskeletal dynamics during cell movement require not only PKA activity, but the specific localization of PKA activity. Currently, there little information regarding the role of spatial regulation of PKA in controlling cytoskeletal dynamics during neurite outgrowth and growth cone motility. Our preliminary evidence strongly suggests that PKA is indeed localized to specific regions within neuronal growth cones. Therefore, we propose to investigate the mechanism and consequences of PKA localization during neurite outgrowth by determining the AKAPs responsible for localization and the cytoskeletal targets within the growth cone affected by PKA. For these studies, a compliment of microscopy of fixed and live cells and biochemical fractionation of neurons into growth cones and soma will provide detailed information on the dynamic distribution of PKA subunits and activity within protrusive neuronal processes. These efforts will provide important insights into the mechanism through which signals from cell adhesion and from neurotrophic factors are coordinated to regulate growth cone guidance.
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