ILK-Actopaxin Interactions in Cell Signaling

描述（由申请人提供）：整合素介导的细胞相互作用 细胞外基质控制着一系列生理过程 包括胚胎发生和转移过程中的细胞迁移， 增殖、组织维持、分化和修复。蛋白 与整联蛋白胞质结构域相关的复合物促进整联蛋白 信号传导和与肌动蛋白细胞骨架的相互作用。这项建议会 重点关注最近确定的 整合素连接激酶ILK、肌动蛋白结合蛋白actopaxin和 分子衔接蛋白桩蛋白在细胞粘附调节中的作用， 运动、增殖和分化。In Aim 1表位标记突变体 将ILK和actopaxin的cDNA转染到成纤维细胞中，然后将其转染到成纤维细胞中。 进一步表征ILK和actopaxin的共沉淀实验 结合域。这些结构域对于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 表明actopaxin突变体干扰细胞粘附/铺展， 胶原目标2中提出的实验将确定以下方面的分子基础： 通过测定整合素功能的变化以及对 下游信号事件。ILK和actopaxin在介导细胞凋亡中的作用 将使用延时显微镜、Boyden室和 伤口分析在目标3中，我们将研究ILK-actopaxin的重要性， 在调节细胞增殖和分化中的关联。位点 相关细胞周期蛋白B/cdc 2激酶导致的actopaxin磷酸化 活动将被描述。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 将通过共沉淀分析检查通过有丝分裂的过渡 和免疫荧光显微术。对增殖和细胞的潜在影响 将检查存活率。ILK和actopaxin相互作用参与 调节细胞分化将在骨骼肌的背景下进行检查， 肌成肌细胞分化这些实验将共同解决 进化上保守的机制，蛋白质之间的联系， 细胞外基质和肌动蛋白细胞骨架，这是重要的， 了解心血管和肌肉骨骼缺陷以及转移性 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.