Molecular Analysis of Signal Transduction in Cell Migration

拟议研究的长期目标是了解脑出血患者体内的信号转导机制。 对细胞迁移的调节。上一期资助的重点是粘着斑激酶(FAK)和 其下游靶标Grb7和一种可能的新型FAK抑制剂FIP200在调节细胞迁移中的作用。我们 鉴定和表征Grb7通过其PH结构域与肌醇磷脂的相互作用 Grb7在介导EphB1刺激细胞迁移中，显示了焦点接触的关键重要性 FAK信号复合体的定位，以及FIP200对FAK活性和细胞的抑制作用 迁移。我们还发现了FAK和N-WASP之间的相互作用，并表明FAK的磷酸化 N-WASP调节N-WASP的亚细胞定位和促进细胞迁移。另外，我们发现， 整合素p1与14-3-3p的相互作用及14-3-3p在细胞调控中的作用 扩散和迁移，以及N-末端FERM样结构域和 FAK的激活域，以一种自动抑制机制调节FAK的激活。在初步研究中， 我们确定了FAK和内嗜素A2之间的相互作用，并表明内嗜素A2与 通过FAK和FAK/Src的磷酸化调节其与Dynamin的相互作用并影响其 Src转化细胞的侵袭。我们还在创造针对乳腺上皮的FAK 条件基因敲除小鼠研究FAK在乳腺癌中的作用及其与亲内素A2的相互作用 体内转移。最后，为了便于研究细胞在ECM梯度上的定向迁移， 模拟活体条件，我们制造了微流控装置来产生明确和定量的FN 在梯度上显示了细胞的定向迁移，具有新的性质。在这项提案中，我们 将1)确定FAK与内亲素A2相互作用在细胞调控中的机制和作用 迁移和侵袭，2)研究FAK的潜在作用及其与内皮亲和素A2的相互作用 3)分析FAK和其他信号分子在肿瘤转移中的反应和作用。 在定义的FN梯度上定向细胞迁移。这些研究将加深我们对 细胞迁移和侵袭中信号转导的分子机制是细胞迁移和侵袭的关键因素 生物过程，如胚胎发育、伤口愈合和癌症。

The long term goal of the proposed studies is to understand the signal transduction mechanisms in the regulation of cell migration. Previous funding period focused on the role of focal adhesion kinase (FAK) and its downstream target Grb7 and a putative novel FAK inhibitor FIP200 in the regulation of cell migration. We identified and characterized Grb7 interaction with phosphoinositides through its PH domain and a role for Grb7 in mediating EphB1 stimulation of cell migration, showed the critical importance of focal contacts localization of the FAK signaling complexes, and characterized FIP200 inhibition of FAK activity and cell migration. We also found an interaction between FAK and N-WASP and showed that FAK phosphorylation of N-WASP regulates N-WASP subcellular localization and promotion of cell migration. In addition, we found an interaction between integrin p1 and 14-3-3p and studied the role of 14-3-3p in the regulation of cell spreading and migration, and an intra-molecular interaction between the N-terminal FERM-like domain and the kinase domain of FAK that regulate FAK activation in an auto-inhibitory mechanism. In preliminary studies, we identified an interaction between FAK and endophilin A2 and showed that endophilin A2 association with FAK and phosphorylation by FAK/Src complex regulated its interaction with dynamin and affected invasion of Src transformed cells. We are also in the process of creating mammary epithelial-specific FAK conditional knockout mice to study the role of FAK and its interaction with endophilin A2 in breast cancer metastasis in vivo. Lastly, to facilitate studies of directional cell migration on ECMgradients which better mimic in vivo conditions, we fabricated microfluidics devices to generate defined and quantitative FN gradients and showed directional cell migration on the gradients with novel properties. In this proposal, we will 1) determine the mechanism and role of FAK interaction with endophilin A2 in the regulation of cell migration and invasion, 2) investigate the potential role of FAK and its interaction with endophilin A2 in cancer metastasis in vivo, and 3) analyze the responses and roles of FAK and other signaling molecules in directional cell migration on defined FN gradients. These studies will enhance our understanding of the molecular mechanisms of signal transduction in cell migration and invasion which are critical factors in biological processes such as embryonic development, wound healing and cancer.