Spatial regulation of Protein Kinase A in cell migration

描述(由申请人提供)：由肌动蛋白细胞骨架动力学驱动的细胞迁移是一种基本的重要细胞行为。正常的细胞迁移程序有助于生物体的发育和动态平衡，而异常迁移是恶性肿瘤转移扩散等病理过程的核心。长期以来，cAMP依赖的蛋白激酶(PKA)在细胞迁移和细胞骨架组织中既有积极作用，也有消极作用。然而，几乎没有工作来协调这些不同的观察结果，并阐明这种古老的和有影响力的激酶对调节细胞迁移的贡献。初步研究表明，对趋化细胞迁移起重要作用的不仅是PKA的活性，而且是该活性的亚细胞分布。具体地说，PKA调节亚基、PKA活性和PKA关键细胞骨架底物的磷酸化在迁移细胞前沿形成的突起结构中显著丰富。此外，抑制PKA活性和破坏PKA定位都会抑制前沿结构的形成和趋化细胞的迁移。这支持了一种假说，即PKA特定地在迁移细胞的前沿被激活，这种局部活动调控着肌动蛋白细胞骨架动力学和细胞迁移的关键调节因子。这项拟议的工作将通过提出两个广泛的问题来检验这一假设：这种定位是如何发生的，以及它的分子后果是什么。结合显微镜(3-、4-和5-D成像；FRET)和生化技术(免疫分析、激酶分析、二维凝胶电泳法、质谱学)将被用来确定PKA定位到前沿的细胞动力学，并确定负责它的锚定蛋白(特定目标1)，以及确定定位的PKA信号对已知的PKA细胞骨架目标的影响，并确定PKA可能调节细胞运动的新靶点(特定目标2)。

DESCRIPTION (provided by applicant): Cell migration, driven by actin cytoskeletal dynamics, is a fundamentally important cellular behavior. Normal programs of cell migration contribute to organism development and homeostasis, while abnormal migration lays at very heart of pathological processes such as the metastatic spread of malignant tumors. The cAMP-dependent protein kinase (PKA) has oft and long been shown to exert both negative and positive effects on cell migration and cytoskeletal organization. However, little work has been done to reconcile these disparate observations and elucidate the contribution of this venerable and influential kinase to the regulation of cell migration. Preliminary studies show that it is not only the activity of PKA, but also the subcellular distribution of that activity, that is important for chemotactic cell migration. Specifically, PKA regulatory subunits, PKA activity, and the phosphorylation of key cytoskeletal substrates for PKA are significantly enriched in protrusive structures formed at the leading edge of migrating cells. Moreover, both inhibition of PKA activity and disruption of PKA localization inhibit formation of leading edge structures and chemotactic cell migration. This supports an hypothesis in which PKA is activated specifically in the leading edge of migrating cells and this localized activity modulates key regulators of actin cytoskeletal dynamics and cell migration. The proposed work will test this hypothesis by asking two broad questions: How does this localization occur and what are its molecular consequences. A combination of microscopy (3-, 4-, and 5-D imaging; FRET) and biochemical techniques (immunoanalyses, kinase assays, 2-D gel electrophoresis; mass spectrometry) will be used to determine the cellular dynamics of PKA localization to the leading edge and identify the anchoring proteins responsible for it (Specific Aim 1), as well as determine the effects of localized PKA signaling on known cytoskeletal targets for PKA and identify new targets through which PKA might regulate cell motility (Specific Aim 2).