FUNCTIONAL STUDIES OF FOCAL ADHESION KINASE

粘着斑激酶的功能研究

• 批准号: 2468101
• 负责人: Steven K. Hanks
• 金额: $ 24.69万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-12-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2468101
• 关键词: animal tissue; biological signal transduction; cell migration; enzyme activity; enzyme induction /repression; extracellular matrix; fibroblasts; focal adhesion kinase; gene mutation; genetic mapping; integrins; laboratory mouse; monoclonal antibody; phosphorylation; protein structure; serine; tissue /cell culture; tyrosine

DESCRIPTION: Integrin-mediated adhesion to the extracellular matrix (ECM) profoundly affect cell shape, motility, survival, proliferation, and differentiation. These behavioral properties are critical for normal growth and development, and impact upon many human pathological processes including tumor growth and metastasis, atherogenesis, thrombosis and hemostasis, inflammation, and cutaneous wound repair. The broad long-term objective of this project is to understand the biochemical signaling mechanisms by which ligand-engaged integrins influence cell behavior, thereby revealing new therapeutic targets for the treatment of adhesion-influenced disease processes. The three specific aims address the general hypothesis that the focal adhesion tyrosine kinase "FAK" is a key signaling molecule affecting integrin-mediated changes in cell behavior. Aim 1 will determine the regulatory mechanisms involved in a FAK-enhanced cell migration response. The effect of inducible FAK expression on the ability of cells to migrate toward defined serum components in a Boyden assay will be assessed, and the FAK functional properties required for the response will be examined by measuring migration rates of cells expressing FAK mutants deficient in specific signaling activities. Aim 2 will examine the role of FAK kinase domain activation loop phosphorylation in integrin signaling responses. The mechanism of FAK activation will be defined by determining the ability of mutant FAK variants, deficient in either activation loop phosphorylation or interactions with Src family kinases, to become fully phosphorylated and catalytically active in response to call adhesion. Monoclonal antibodies that specifically recognize the phosphorylated FAK activation loop will be employed to reveal the dynamics and regionalization of activated FAK during the course of cell spreading and migration. Aim 3 will examine the role of serine phosphorylation on FAK's signaling properties and adhesion-stimulated cellular responses. The major site of FAK serine phosphorylation in adherent fibroblasts will be determined by a phosphopeptide mapping strategy, and the potential role of this phosphorylation event in regulating FAK's signaling capacity and functional properties will be assessed through expression and analysis of the phosphoaccentor serine FAK mutant.

描述：整合素介导的细胞外基质 (ECM) 粘附 深刻影响细胞的形状、运动、存活、增殖和 差异化。 这些行为特性对于正常生长至关重要 和发展，以及对许多人类病理过程的影响，包括 肿瘤生长和转移、动脉粥样硬化、血栓形成和止血， 炎症和皮肤伤口修复。 广泛的长期目标 该项目旨在了解生化信号传导机制 配体结合的整合素影响细胞行为，从而揭示新的 治疗粘连影响疾病的治疗靶标 流程。 这三个具体目标解决了以下一般假设： 粘着斑酪氨酸激酶“FAK”是影响粘着斑的关键信号分子 整合素介导的细胞行为变化。 目标 1 将确定 FAK 增强的细胞迁移反应涉及的调节机制。 诱导型FAK表达对细胞迁移能力的影响 将评估博伊登测定中确定的血清成分，并且 响应所需的 FAK 功能属性将由以下人员检查 测量表达FAK突变体的细胞的迁移率 特定的信号活动。 目标 2 将检查 FAK 激酶的作用 整合素信号传导反应中的结构域激活环磷酸化。 这 FAK激活的机制将通过确定的能力来定义 突变的 FAK 变体，缺乏激活环磷酸化或 与 Src 家族激酶相互作用，使其完全磷酸化并 对呼叫粘附具有催化活性。 单克隆抗体 特异性识别磷酸化 FAK 激活环的 用于揭示激活的 FAK 的动态和区域化 细胞扩散和迁移的过程。 目标 3 将检验以下角色的作用： 丝氨酸磷酸化对 FAK 信号传导特性和粘附刺激的影响 细胞反应。 FAK 丝氨酸磷酸化的主要位点 贴壁成纤维细胞将通过磷酸肽图谱确定 策略，以及该磷酸化事件在调节中的潜在作用 FAK 的信号传输能力和功能特性将通过以下方式进行评估： 磷酸重音中心丝氨酸FAK突变体的表达和分析。