ILK-Actopaxin Interactions in Cell Signaling

描述(申请人提供)：整合素介导的细胞相互作用 细胞外基质控制着一系列不同的生理过程 包括胚胎发生和转移过程中的细胞迁移，细胞 增殖、组织维持、分化和修复。蛋白 与整合素胞质结构域相关的复合体促进整合素 肌动蛋白细胞骨架的信号和相互作用。这项提议将 重点关注最近发现的 整合素连接的激酶ILK、肌动蛋白结合蛋白actopaxin和 分子适配蛋白Paxlin在调节细胞黏附中的作用 运动性、增殖性和分化。在AIM 1表位标记突变体中 ILK和actopaxin的cDNA将被导入成纤维细胞，随后 共沉淀实验进一步表征ILK和Actopaxin 结合域。这些结构域对亚细胞定位的重要性 将通过免疫荧光显微镜进行评估。我们最近做了 证明Actopaxin突变体扰乱细胞黏附/扩散 胶原蛋白。在目标2中提出的实验将确定分子基础 这种缺陷是通过分析整合素功能的变化以及对 下行信令事件。ILK和actopaxin在细胞中介中的作用 运动将使用延时显微镜，博伊登小室和 伤口化验。在目标3中，我们将研究ILK-actopaxin的重要性 调节细胞增殖和分化的关联。网站： 相关细胞周期蛋白B/cdc2激酶导致的actopaxin磷酸化 活动将被描绘出来。这些位点的磷酸化在 调节蛋白质-蛋白质相互作用和相关的细胞骨架变化 通过有丝分裂的过渡将通过共沉淀分析进行检测 和免疫荧光显微镜。对增殖和细胞的潜在影响 存活率将会被检查。ILK和actopaxin的相互作用参与血管内皮细胞瘤的发生 调节细胞分化将在骨骼的背景下进行研究 肌成肌细胞分化。这些实验将共同解决 进化上保守的蛋白质连接机制 细胞外基质和肌动蛋白细胞骨架对 对心血管和肌肉骨骼缺陷及转移的认识 转型。

DESCRIPTION (provided by applicant): Integrin-mediated interaction of cells with the extracellular matrix control a diverse set of physiologic processes including cell migration during embryogenesis and metastasis, cell proliferation, tissue maintenance, differentiation and repair. Protein complexes associated with the integrin cytoplasmic domains facilitate integrin signaling and interactions with the actin cytoskeleton. This proposal will focus on the importance of recently identified interactions between the integrin-linked kinase ILK, the actin binding protein actopaxin and the molecular adapter protein paxillin in the regulation of cell adhesion, motility, proliferation and differentiation. In Aim 1 epitope-tagged mutant cDNAs of ILK and actopaxin will be transfected into fibroblasts followed by co-precipitation experiments to further characterize the ILK and actopaxin binding domains. The importance of these domains for subcellular localization will be assessed by immunofluorescence microscopy. We have recently demonstrated that actopaxin mutants perturb cell adhesion/spreading on collagen. Experiments proposed in Aim 2 will identify the molecular basis for this defect by assaying for changes in integrin function as well as effect on downstream signaling events. The role of ILK and actopaxin in mediating cell motility will be addressed using time-lapse microscopy, Boyden chamber and wound assays. In Aim 3 we will investigate the importance of ILK-actopaxin associations in regulating cell proliferation and differentiation. Sites of actopaxin phosphorylation resulting from associated cyclinB/cdc2 kinase activity will be delineated. The role of phosphorylation of these sites in regulating protein-protein interactions and cytoskeleton changes associated with transition through mitosis will be examined by co-precipitation analysis and immunofluorescence microscopy. Potential effects on proliferation and cell survival will be examined. The involvement of ILK and actopaxin interactions in regulating cell differentiation will be examined in the context of skeletal muscle myoblast differentiation. Together these experiments will address evolutionarily conserved mechanisms of protein linkages between the extracellular matrix and the actin cytoskeleton that are of importance to the understanding of cardiovascular and musculoskeletal defects and metastatic transformation.