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体内平衡，而异常的细胞迁移是病理过程的核心，如恶性肿瘤的转移扩散。camp依赖性蛋白激酶（PKA）长期以来一直被证明对细胞迁移和细胞骨架组织有积极和消极的影响。然而，很少有研究能够调和这些不同的观察结果，并阐明这种古老而有影响力的激酶对细胞迁移调节的贡献。初步研究表明，PKA的活性及其亚细胞分布是趋化细胞迁移的重要因素。具体来说，PKA调控亚基、PKA活性以及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富集。此外，PKA活性的抑制和PKA定位的破坏都会抑制前沿结构的形成和趋化细胞的迁移。这支持了一种假设，即PKA在迁移细胞的前沿被特异性激活，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验证这一假设：这种定位是如何发生的，以及它的分子后果是什么。显微术（3-、4-和5-D成像；FRET）和生化技术(免疫分析、激酶测定、二维凝胶电泳；质谱法)将用于确定PKA定位到前沿的细胞动力学，并确定负责它的锚定蛋白（Specific Aim 1），以及确定PKA信号定位对已知PKA细胞骨架靶点的影响，并确定PKA可能通过其调节细胞运动的新靶点（Specific Aim 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).