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

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心机构，不一定是研究者机构。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，肌动蛋白细胞骨架动力学响应于吸引或排斥的引导线索。重要的是，生长锥吸引和排斥之间的平衡由cAMP和cAMP依赖性蛋白激酶（PKA）调节。我们实验室最近的研究结果表明PKA以一种非常有趣的方式主持成纤维细胞的趋化迁移。具体而言，PKA调节亚基的子集，以及PKA活性，和磷酸化的细胞骨架的目标都显着丰富的突出伪足和板状伪足。此外，抑制PKA活性和抑制PKA与AKAP（A-激酶锚定蛋白）的相互作用防止伪足形成并抑制趋化性。这表明，在细胞运动过程中形成的膨胀结构不仅需要PKA活性，但PKA活性的特定定位。虽然AKAP介导的PKA定位已被证明是重要的神经元功能的某些方面，尽管PKA信号调节生长锥的指导的核心重要性，有很少或没有信息的作用，空间调节PKA的轴突生长和生长锥运动。因此，我们建议确定PKA是否在轴突生长过程中受到空间调节，并研究AKAP介导的PKA定位是否是有效的轴突生长和轴突导向所必需的。对于这些研究，一个恭维的固定和活细胞的显微镜和生物化学分馏的生长锥和轴突小丘PKA亚基的动态分布和活动的详细信息，在神经元的过程。这些努力将提供重要的初步数据，可能涉及PKA活性的空间调节作为一个重要的，迄今为止尚未调查的生长锥指导方面。

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.