PP1-SPATIAL REGULATION OF PROTEIN KINASE A SIGNALING DURING GROWTH CONE GUIDANCE

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。神经元生长锥的突起、运动和引导是由肌动蛋白细胞骨架动力学驱动的，肌动蛋白对吸引或排斥的引导信号做出反应。重要的是，生长锥吸引和排斥之间的平衡是由cAMP和cAMP依赖性蛋白激酶（PKA）调节的。我们实验室最近的结果表明，PKA以一种最有趣的方式主持成纤维细胞的趋化迁移。具体来说，PKA调控亚基的一个子集，以及PKA活性和一些细胞骨架靶点的磷酸化都在突出的假足和板足中显著富集。此外，抑制PKA活性和抑制PKA与AKAPs （a -激酶锚定蛋白）的相互作用可阻止假足的形成并抑制趋化性。这表明细胞运动过程中突起结构的形成不仅需要PKA活性，还需要PKA活性的特定定位。尽管akap介导的PKA定位已被证明对神经元功能的某些方面很重要，尽管PKA信号在调节生长锥引导方面具有核心重要性，但关于PKA在神经突生长和生长锥运动中的空间调节作用的信息很少或没有。因此，我们建议确定PKA是否在神经突生长过程中受到空间调控，并研究akap介导的PKA定位是否需要有效的神经突生长和轴突引导。在这些研究中，固定细胞和活细胞的显微镜观察以及生长锥和轴突丘的生化分离将提供PKA亚基动态分布和突起神经元过程活动的详细信息。这些努力将提供重要的初步数据，可能暗示PKA活性的空间调节是生长锥指导的一个重要且迄今未被研究的方面。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The protrusion, movement and guidance of neuronal growth cones are driven by actin cytoskeletal dynamics that respond to attractive or repulsive guidance cues. Importantly, the balance between growth cone attraction and repulsion is modulated by cAMP and the cAMP-dependent protein kinase (PKA). Recent results from our laboratory have shown that PKA presides over the chemotactic migration of fibroblasts in a most intriguing way. Specifically, 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. Moreover, both inhibition of PKA activity and inhibition of PKA interaction with AKAPs (A-kinase anchoring proteins) prevent pseudopod formation and inhibit chemotaxis. This suggests that the formation of protrusive structures during cell movement requires not only PKA activity, but the specific localization of PKA activity. Although AKAP-mediated PKA localization has been shown to be important for some aspects of neuronal function, and despite the central importance of PKA signaling for regulating growth cone guidance, there is little to no information regarding the role of spatial regulation of PKA in neurite outgrowth and growth cone motility. Therefore, we propose to determine whether PKA is spatially regulated during neurite outgrowth and to investigate whether AKAP-mediated localization of PKA is required for efficient neurite outgrowth and axon guidance. For these studies, a compliment of microscopy of fixed and live cells and biochemical fractionation of growth cones and axon hillocks will provide detailed information on the dynamic distribution of PKA subunits and activity within protrusive neuronal processes. These efforts will provide important preliminary data that may implicate spatial regulation of PKA activity as an important and heretofore uninvestigated facet of growth cone guidance.