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非常重要的细胞行为。正常的细胞迁移程序有助于生物体的发育和稳态，而异常的迁移则是恶性肿瘤转移扩散等病理过程的核心。cAMP依赖性蛋白激酶（PKA）对细胞迁移和细胞骨架的形成有积极和消极的作用。然而，很少有工作已经做了调和这些不同的意见，并阐明了这一古老的和有影响力的激酶的调节细胞迁移的贡献。初步研究表明，PKA的活性及其亚细胞分布对趋化细胞迁移具有重要作用。具体而言，PKA调节亚基，PKA活性和PKA的关键细胞骨架底物的磷酸化显着富集在迁移细胞的前缘形成的突起结构。此外，PKA活性的抑制和PKA定位的破坏都抑制前缘结构的形成和趋化细胞迁移。这支持了PKA在迁移细胞的前沿被特异性激活的假设，并且这种局部活性调节肌动蛋白细胞骨架动力学和细胞迁移的关键调节因子。拟议的工作将通过提出两个广泛的问题来测试这一假设：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）和生物化学技术（免疫分析、激酶测定、2-D凝胶电泳;质谱）将被用来确定PKA定位到前沿的细胞动力学，并鉴定负责它的锚定蛋白（具体目标1），以及确定PKA信号传导对PKA的已知细胞骨架靶点的影响，并确定PKA可能调节细胞运动性的新靶点（具体目标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).