FAK/Src/CAS Signaling in Cell Motility and Invasion

描述(申请人提供)：摘要。细胞-细胞外基质黏附中蛋白质的酪氨酸磷酸化是细胞增殖、运动和侵袭的主要因素。整合素介导的黏附激活由两个酪氨酸激酶：FAK和Src结合而形成的信号复合体。FAK/Src复合体的一个主要底物是对接蛋白p130Cas，它在其“底物结构域”上经历了大量的酪氨酸残基的磷酸化。最近的研究表明，FAK/Src/CAS复合信号在细胞运动和侵袭的相关过程中具有重要作用。该项目的长期目标是充分了解FAK/Src/CAS复合体激活所需的分子相互作用，以及下游信号事件如何影响细胞运动和入侵的动态事件。提出了三个具体目标。AIM 1将使用荧光标记的CAS变体的活细胞成像来确定CAS黏附位点靶向的动力学和机制。对CAS相互作用蛋白的鉴定对其定位也很重要。目的2将利用时间推移显微镜来确定CAS底物结构域磷酸化对驱动细胞运动的片状脂膜突起和黏附组装/解体的动态过程以及驱动入侵的足体-内足的动态组装的影响。在相关研究中，将研究CAS底物结构域磷酸化促进肌动蛋白聚合的能力。虽然前两个AIM使用的是CAS缺失的小鼠成纤维细胞模型，但Aim 3通过使用RNA干扰干扰CAS表达来扩展对侵袭性人类乳腺癌细胞系的建模工作。将评估CAS基因敲除对体外细胞增殖和侵袭以及对体内肿瘤生长和转移的影响。野生型与CAS突变变体的重组实验将揭示重要的功能结构域。关联性。拟议中的研究将填补我们对基本信号机制理解的关键空白，这些基本信号机制驱动着对细胞运动和入侵至关重要的复杂动态事件。这些事件在正常的组织发育/再生以及许多病理过程中起着核心作用，包括关节炎、动脉粥样硬化、炎症、骨质疏松症和癌症进展。这项工作有可能揭示由异常运动和侵袭引起的疾病的新的治疗靶点和预后指标。

DESCRIPTION (provided by applicant): Summary. Tyrosine phosphorylation of proteins in cell-ECM adhesions is a major factor driving the proliferative growth, motility, and invasion of cells. Integrin-mediated adhesion activates a signaling complex formed by the association of two tyrosine kinases: FAK and Src. A major substrate of the FAK/Src complex is the docking protein p130CAS, which undergoes phosphorylation on numerous tyrosine residues in its "substrate domain". Recent studies demonstrated the importance of FAK/Src/CAS complex signaling in the related processes of cell motility and invasion. The broad long-term objective of this project is to fully understand the molecular interactions necessary for FAK/Src/CAS complex activation and how downstream signaling events impact the dynamic events that underlie cell motility and invasion. Three specific aims are proposed. Aim 1 will use live cell imaging of fluorescent-tagged CAS variants to determine the dynamics and mechanism of CAS adhesion site targeting. The identification of CAS-interacting proteins important for its localization will also be pursued. Aim 2 will employ time-lapse microscopy to determine the impact of CAS substrate domain phosphorylation on the dynamic processes of lamellipodial protrusion and adhesion assembly/disassembly that drive cell motility, and on the dynamic assembly of podosomes-invadopodia that drive invasion. In related studies, the capacity of CAS substrate domain phosphorylation to promote actin polymerization will be investigated. While the first two Aims make use of CAS-null mouse fibroblast models, Aim 3 extends the work to model invasive human breast cancer cell lines through the use RNA interference to disrupt CAS expression. The impact of the CAS knockdown on cell proliferation and invasion in vitro and on tumor growth and metastasis in vivo will be assessed. Reconstitution experiments with wild-type vs. CAS mutational variants will reveal the important functional domains. Relevance. The proposed studies will fill key gaps in our understanding of the basic signaling machinery that drives the complex dynamic events important for cell motility and invasion. These events have a central role in normal tissue development/regeneration and in many pathological processes including arthritis, atherosclerosis, inflammation, osteoporosis, and cancer progression. The work has potential to reveal new therapeutic targets and prognostic indicators for diseases that arise from aberrant motility and invasion.