FUNCTIONAL STUDIES OF FOCAL ADHESION KINASE

细胞与细胞外基质（ECM）的相互作用深刻地影响着 细胞形状、迁移、增殖和分化-特性 这对正常的生长和发育很重要， 健康护理和疾病的许多方面包括伤口修复， 血管生成、癌症转移和动脉粥样硬化形成。 的机制 ECM对细胞行为的影响仍然是一个基本的未解决的问题， 细胞生物学的问题。最近的证据表明，酪氨酸- 蛋白质的磷酸化是应答中的关键事件。支持 这是一种新的酪氨酸激酶，已被鉴定并显示为 定位于"粘着灶"，即与ECM相互作用的部位 通过整合素受体发生。进一步的研究表明，这种激酶， 称为"粘着斑激酶"（FAK），变得高度磷酸化 (on酪氨酸和丝氨酸位点）， 结合整联蛋白受体的其他ECM底物。这些 磷酸化事件可能是整合素介导的 参与FAK的信号通路。 拟议工作的长期目标是全面实现 理解FAK在实现受ECM影响的变化中的作用 在细胞行为中。具体来说，FAK磷酸化的主要位点诱导 将测定小鼠成纤维细胞对纤连蛋白的粘附 通过磷酸肽作图策略，以及这些策略的效果， 对FAK激酶活性的修饰，粘着斑靶向， 将评价与已知SH 2-蛋白相互作用的能力。获得 为了进一步了解FAK的信号作用，两种分子方法将 用于鉴定与FAK相互作用的蛋白质。一种方法 目的是鉴定与FAK相互作用的新型SH2蛋白 磷酸酪氨酸另一种方法涉及强大的基因筛选 寻找能与FAK分子任何部分稳定相互作用的蛋白质。 这些蛋白质可能作为上游调节剂， FAK的下游效应子。

Cell interactions with the extracellular matrix (ECM) profoundly influence cell shape, migration, proliferation and differentiation - properties which are important for normal growth and development, and which affect many aspects of health care and disease including wound repair, angiogenesis, cancer metastasis, and atherogenesis. The mechanism by which the ECM influences cell behavior remains one of the basic unsolved problems in cell biology today. Recent evidence indicates that tyrosine- phosphorylation of proteins is a key event in the response. In support of this, a novel tyrosine kinase has been identified and shown to be localized to "focal adhesions," sites where interactions with the ECM occur via integrin receptors. Further studies indicate that this kinase, designated "Focal Adhesion Kinase" (FAK), becomes highly phosphorylated (on both tyrosine and serine sites) upon cell adhesion to fibronectin and other ECM substrates that bind to integrin receptors. These phosphorylation events are likely to be essential to an integrin-mediated signaling pathway involving FAK. The long term objective of the proposed work is to reach a full understanding of the role of FAK in bringing about ECM-influenced changes in cell behavior. Specifically, major sites of FAK phosphorylation induced upon the adherence of mouse fibroblasts to fibronectin will be determined by phosphopeptide mapping strategies, and the effect of these modifications on FAK's kinase activity, focal adhesion targeting, and ability to interact with known SH2-proteins will be evaluated. To gain further insight into FAK's signaling role, two molecular approaches will be undertaken to identify proteins that interact with FAK. One approach aims to identify novel SH2 proteins that interact with FAK phosphotyrosines. The other approach involves a powerful genetic screen for proteins that can stably interact with any part of the FAK molecule. Such proteins are likely to act as either upstream regulators or downstream effectors of FAK.