Protein Kinase A in Focal Adhesions - Mechanisms and Consequences

项目摘要 细胞与周围细胞外基质（ECM）的相互作用控制着几乎所有主要的细胞功能 - 包括生长、分裂、生存、形状和运动。ECM间接连接到 粘着斑处的细胞内肌动蛋白细胞骨架（FA）-组装并 肌动蛋白微丝被分解以动态地偶联到基质结合的整联蛋白的细胞质尾部。在 除了它们的耦合功能，FA还发送和接收信号，以传达和控制粘合剂 细胞的状态。在这些信号中突出的是可逆的蛋白质磷酸化，其中蛋白质含有 以及控制磷酸酪氨酸在FA生物学中特别丰富、重要和研究充分。这 尽管重要，FA蛋白的磷酸丝氨酸和磷酸苏氨酸修饰远比 比磷酸酪氨酸丰富，但研究和理解的少得多。 蛋白激酶A（Protein Kinase A，PKA）是一种广泛存在的丝氨酸/苏氨酸激酶，在细胞内具有复杂的调节作用 迁移一些观察结果表明，PKA也可能是重要的信号内脂肪酸。第一，抑制 PKA在细胞附着早期改变FA动力学，增加FA大小和聚集，并减少细胞粘附。 蔓延此外，PKA亚基以及一些推定的PKA底物已被鉴定， 分离的粘附复合物以及已发表的整合素和/或粘着斑相关蛋白质组。 此外，PKA亚基以及一些已建立和推定的PKA底物已被确定 在分离的粘附复合物和已发表的整合素和/或粘着斑相关蛋白质组中。使用 焦点粘附靶向PKA生物传感器，我们最近已经显示出高度本地化和动态PKA活性内 单个局灶性粘连。最后，使用邻近标记和互补生物化学方法，我们 已经表明PKA调节（R）亚基与原型粘着斑蛋白talin密切相互作用。我们 还报道PKA磷酸化talin以及talin相关蛋白（TAP）RIAM、DLC 1和TES。 基于这些观察，我们假设PKA的离散池与talin相互作用，修饰talin， TAPs，并增强FA动态。 总之，PKA与talin相关，但这种相互作用的生化性质和决定因素是 不知道--这是具体目标1的目标。PKA似乎直接磷酸化talin，但位点和 这种修改的效果尚不清楚-这是具体目标2的目标。PKA似乎直接磷酸化 几种talin相关蛋白，但这些修饰的位点和作用尚不清楚-这是 具体目标3。最后，虽然talin和TAP似乎代表PKA的重要靶簇， 调节这一簇对细胞粘附、FA动力学、细胞扩散和迁移的影响并不 这是第四个明确的目标。

PROJECT SUMMARY Cell interaction with the surrounding extracellular matrix (ECM) controls nearly every major cellular function – including growth, division, survival, shape, and movement. The ECM is connected, vicariously, to the intracellular actin cytoskeleton at focal adhesions (FAs) – multi-protein complexes that assemble and disassemble to dynamically couple actin microfilaments to the cytoplasmic tails of matrix-bound integrins. In addition to their coupling function, FAs also send & receive signals that communicate & control the adhesive state of the cell. Prominent among these signals is reversible protein phosphorylation, with proteins containing and controlling phosphotyrosine being particularly abundant, important, and well-studied in FA biology. This importance notwithstanding, phosphoserine and phosphothreonine modification of FA proteins is far more abundant than phosphotyrosine, but far less studied and understood. Protein Kinase A (PKA) is a ubiquitous and promiscuous Ser/Thr kinase with complex regulatory roles in cell migration. Several observations suggest that PKA may also be important for signaling within FAs. First, inhibition of PKA early during cell attachment alters FA dynamics, increasing FA size and clustering, and decreases cell spreading. Furthermore, PKA subunits as well as a number of putative PKA substrates have been identified in isolated adhesion complexes as well as in published integrin- and/or focal adhesion-associated proteomes. Furthermore, PKA subunits as well as a number of established and putative PKA substrates have been identified in isolated adhesion complexes and in published integrin- and/or focal adhesion-associated proteomes. Using a focal adhesion-targeted PKA biosensor, we have recently shown highly localized and dynamic PKA activity within individual focal adhesions. Finally, using proximity-labelling and complimentary biochemical approaches, we have shown that PKA regulatory (R) subunits closely interact with the archetypal focal adhesion protein talin. We also report that PKA phosphorylates talin as well as the talin-associated proteins (TAPs) RIAM, DLC1, and TES. Based on these observations, we hypothesize that a discrete pool of PKA interacts with talin, modifies talin and TAPs, and enhances FA dynamics. In summary, PKA associates with talin, but the biochemical nature and determinants of this interaction are not known – this is the goal of Specific Aim 1. PKA appears to directly phosphorylate talin, but the sites and effects of this modification are not known - this is goal of Specific Aim 2. PKA appears to directly phosphorylate several talin-associated proteins, but the sites and effects of these modifications are not known – this is the goal of Specific Aim 3. Finally, while talin and TAPs appear to represent an important target cluster for PKA, the consequences of regulating this cluster on cell adhesion, FA dynamics, cell spreading and migration are not known – this is the goal of Specific Aim 4.