Protein Kinase A in Focal Adhesions - Mechanisms and Consequences

局灶性粘连中的蛋白激酶 A - 机制和后果

• 批准号: 10156931
• 负责人: Alan K Howe
• 金额: $ 37.54万
• 依托单位: UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-10 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10156931
• 关键词: A kinase anchoring protein; Actins; Actomyosin; Adhesions; Adhesives; Behavior; Binding; Biochemical; Biology; Biosensor; Calpain; Cell Adhesion; Cell Communication; Cell physiology; Cell-Matrix Junction; Cells; Complex; Coupling; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Tail; Cytoskeleton; DLEC1 gene; Data; Environment; Enzymes; Extracellular Matrix; Focal Adhesions; Goals; Growth; In Vitro; Individual; Integrin Binding; Integrins; Investigation; Label; Learning; Maps; Mechanics; Mediating; Microfilaments; Modification; Movement; Nature; Persons; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphoserine; Phosphothreonine; Phosphotransferases; Phosphotyrosine; Predisposition; Protein Inhibition; Proteins; Proteome; Publishing; Regulation; Reporter; Reporting; Resistance; Role; Sampling; Shapes; Signal Transduction; Signaling Protein; Site; Specific qualifier value; Structure; Subcellular Spaces; Talin; Testing; base; cell motility; in vivo; insight; migration; mimetics; mutant; protein complex; protein function; scaffold

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.

项目概要 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 间接地与身体相连。 粘着斑 (FA) 处的细胞内肌动蛋白细胞骨架 – 组装和组装的多蛋白复合物 分解以动态地将肌动蛋白微丝耦合到基质结合的整联蛋白的细胞质尾部。 除了耦合功能外，FA 还发送和接收信号来通信和控制粘合剂 这些信号中最突出的是可逆的蛋白质磷酸化，其中蛋白质含有 控制磷酸酪氨酸在 FA 生物学中特别丰富、重要且得到充分研究。 尽管很重要，但 FA 蛋白的磷酸丝氨酸和磷酸苏氨酸修饰更为重要 比磷酸酪氨酸丰富，但研究和理解却少得多。 蛋白激酶 A (PKA) 是一种普遍存在且混杂的 Ser/Thr 激酶，在细胞中具有复杂的调节作用 一些观察结果表明 PKA 对 FA 内的信号传导也很重要。 细胞附着早期的 PKA 改变 FA 动力学，增加 FA 大小和聚集，并减少细胞 此外，PKA 亚基以及许多假定的 PKA 底物已在 分离的粘附复合物以及已发表的整合素和/或粘附斑相关蛋白质组。 此外，PKA 亚基以及许多已确定的和推定的 PKA 底物已被鉴定 使用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。 粘着斑靶向 PKA 生物传感器，我们最近展示了高度局部化和动态的 PKA 活性 最后，我们使用邻近标记和互补生化方法。 我们已经证明 PKA 调节 (R) 亚基与原型粘着斑蛋白 talin 密切相互作用。 还报道 PKA 磷酸化踝蛋白以及踝蛋白相关蛋白 (TAP) RIAM、DLC1 和 TES。 基于这些观察，我们勇敢地发现 PKA 的离散池与 talin 相互作用，修饰 talin 并 TAP，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 未知——这是具体目标 1 的目标。PKA 似乎直接磷酸化 talin，但位点和 这种修饰的效果尚不清楚 - 这是特定目标 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 的目标。最后，虽然 talin 和 TAP 似乎代表了 PKA 的重要目标簇， 调节该簇对细胞粘附、FA 动力学、细胞扩散和迁移的影响并不明显 已知——这是具体目标 4 的目标。