Molecular Analysis Of Signal Transduction by Integrins

描述（改编自申请人的摘要）： 目前的建议是了解信号转导机制， 整合素介导的细胞迁移。我们一直致力于 参与细胞迁移调节的细胞内信号分子。 使用稳定的CHO细胞转染模型，我们鉴定了接头分子 Cas在细胞迁移中作为FAK信号传导的介导剂。此外，我们还展示了 FAK与PI 3 K的相互作用是刺激细胞迁移所必需的， 并且FAK/Src复合物的形成和随后的Cas磷酸化是 还不够。我们还发现FAK与另一个衔接分子相互作用 Grb 7的相互作用，并提供了证据的潜在功能， 调节细胞迁移。进一步的研究表明，Grb 7的募集 与焦点接触及其通过FAK的磷酸化在 Grb 7刺激细胞迁移及其与FAK的相互作用。最后我们 利用酵母克隆了FAK家族激酶的新型蛋白抑制剂FIP 200 FIP 200在体外可抑制FAK活性 和体内。在这个建议中，我们计划通过以下方式确定分子机制： Grb 7和FIP 200参与信号通路的调节， 细胞迁移在目标1中，我们将首先确定关键域和密钥 Grb 7的残基在其调节细胞迁移中的作用 诱变然后，我们将研究这些领域在监管中的作用 Grb 7的磷酸化。最后，我们将直接映射Grb 7 磷酸化位点。在目标2中，我们将研究这些关系， FAK下游信号通路通过FAK/Src/Cas的可能合作， FAK/PI 3 K和FAK/Grb 7复合物。我们将确定亚细胞定位 激活的复合物。我们还将创建和分析FAK的作用 选择性结合这些下游信号分子的突变体。在 最后一个目的是探讨FIP 200的作用机制和调控机制 抑制细胞迁移中的FAK信号传导途径。康贝特人将以 FIP 200的功能结构域使用突变的方法。我们亦会研究 FIP 200抑制FAK下游信号传导途径，使用稳定的可诱导 细胞系，研究内源性FIP 200/FAK的调节和功能 复杂的细胞迁移。这些研究将增进我们对 整合素信号转导的分子机制，这是至关重要的 生物过程中细胞迁移和侵袭的因素， 胚胎发育、伤口愈合和癌症。

DESCRIPTION(adapted from applicant's abstract): The long-term goals of the present proposal are to understand signal transduction mechanisms in integrin-mediated cell migration. We have been focusing on identifying intracellular signaling molecules involved in the regulation of cell migration. Using a stable CHO cell transfection model, we identified the adaptor molecule Cas as a mediator of FAK signaling in cell migration. Furthermore, we showed that FAK interaction with PI3K is required for stimulation of cell migration and that the FAK/Src complex formation and subsequent Cas phosphorylation is not sufficient. We also found FAK interaction with another adaptor molecule Grb7 and provided evidence of potential function of the interaction in the regulation of cell migration. Further studies suggested that Grb7 recruitment to the focal contacts and its phosphorylation by FAK play a role in the stimulation of cell migration by Grb7 and its interaction with FAK. Finally, we have cloned a novel protein inhibitor FIP200 for FAK family kinases using yeast two hybrid screening and showed that FIP200 could inhibit FAK activity in vitro and in vivo. In this proposal, we plan to determine the molecular mechanisms by which Grb7 and FIP200 participate in signaling pathways in the regulation of cell migration. In Aim l, we will first determine the critical domains and key residues of Grb7 in its regulation of cell migration using site-directed mutagenesis. We will then examine the role of these domains in the regulation of Grb7 phosphorylation by FAK. Lastly, we will directly map the Grb7 phosphorylation sites by FAK. In Aim 2, we will examine the relationships and possible cooperations of FAK downstream signaling pathways through FAK/Src/Cas, FAK/PI3K and FAK/Grb7 complexes. We will determine the subcellular localization of the activated complexes. We will also create and analyze the role of FAK mutants with selective binding to these downstream signaling molecules. In the last Aim, we will investigate the mechanisms and regulation of FIP200 inhibition of FAK signaling pathways in cell migration. We will determine the functional domains of FIP200 using mutational approaches. We will also examine FIP200 inhibition of FAK downstream signaling pathways using stable inducible cell lines, study the regulation and functions of the endogenous FIP200/FAK complex in cell migration. These studies will enhance our understanding of the molecular mechanisms of signal transduction by integrins, which are critical factors in cell migration and invasiveness in biological processes such as embryonic development, wound healing and cancer.