FUNCTIONAL STUDIES OF FOCAL ADHESION KINASE

描述：整合素介导的细胞外基质(ECM)黏附 深刻地影响细胞的形状、运动、存活、增殖和 差异化。这些行为特征对正常生长至关重要 并对许多人类病理过程产生影响，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。宏伟的长期目标 这个项目是为了了解生物化学信号机制，通过它 配体参与的整合素影响细胞行为，从而揭示新的 粘附性疾病的治疗靶点 流程。这三个具体目标解决了普遍的假设，即 粘着斑酪氨酸激酶FAK是一个关键的信号分子，影响 整合素介导的细胞行为变化。目标1将决定 FAK增强的细胞迁移反应涉及的调控机制。 可诱导的FAK表达对细胞迁移能力的影响 将对博伊登试验中确定的血清成分进行评估， 将检查响应所需的FAK功能属性 表达FAK突变体的细胞迁移率的测定 特定的信号活动。AIM 2将研究FAK激酶的作用 整合素信号反应中的结构域激活环磷酸化。这个 FAK的激活机制将通过确定其激活能力来确定 FAK突变体，缺乏激活环磷酸化或 与Src家族激酶的相互作用，变得完全磷酸化和 对呼唤粘连的反应具有催化活性。单克隆抗体 具体识别磷酸化FAK激活环的将是 用来揭示激活的FAK在 细胞扩散和迁移的过程。目标3将考察 丝氨酸磷酸化对FAK信号转导特性和黏附刺激的影响 细胞反应。FAK丝氨酸磷酸化的主要部位 贴壁成纤维细胞将通过磷酸肽图谱确定 策略，以及这种磷酸化事件在调控中的潜在作用 FAK的信令容量和功能属性将通过 磷酸重音蛋白丝氨酸FAK突变体的表达和分析。

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.