FAK/Src/CAS Signaling in Cell Motility and Invasion

细胞运动和侵袭中的 FAK/Src/CAS 信号转导

• 批准号: 7907309
• 负责人: Steven K. Hanks
• 金额: $ 18.23万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2011-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7907309
• 关键词: Actins; Adhesions; Ants; Arthritis; Atherosclerosis; Automobile Driving; BCAR1 gene; Behavior; Binding; Breast; Breast Cancer Cell; Breast Carcinoma; C-terminal; Cancer cell line; Cell Line; Cell Proliferation; Cells; Cellular biology; Complex; Development; Disease; Docking; Event; Family; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesions; Growth; Human; In Vitro; Inflammation; Integrins; Knockout Mice; Life; Malignant - descriptor; Malignant Epithelial Cell; Malignant Neoplasms; Mammalian Cell; Mediating; Methods; Microscopy; Modeling; Molecular; Mus; Natural regeneration; Neoplasm Metastasis; Noma; Normal tissue morphology; Osteoporosis; Pathologic Processes; Phosphorylation; Phosphorylation Site; Physiology; Plasma; Process; Property; Protein Tyrosine Kinase; Proteins; RNA Interference; Research; Role; Signal Transduction; Site; Substrate Domain; Testing; Time; Total Internal Reflection Fluorescent; Tyrosine; Tyrosine Phosphorylation; Variant; Work; cell behavior; cell motility; cell type; cellular imaging; frontier; human BCAR1 protein; in vivo; new therapeutic target; polymerization; prognostic indicator; reconstitution; research study; time use; tissue regeneration; tumor growth; tumor progression

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.

描述（由申请人提供）：摘要。细胞-ECM粘附中蛋白质的酪氨酸磷酸化是驱动细胞增殖生长、运动和侵袭的主要因素。整合素介导的粘附激活由两种酪氨酸激酶FAK和Src缔合形成的信号复合物。FAK/Src复合物的主要底物是对接蛋白p130 CAS，其在其“底物结构域”中的许多酪氨酸残基上经历磷酸化。最近的研究表明FAK/Src/CAS复合信号在细胞运动和侵袭相关过程中的重要性。该项目的广泛的长期目标是充分了解FAK/Src/CAS复合物激活所必需的分子相互作用，以及下游信号事件如何影响细胞运动和侵袭的动态事件。提出了三个具体目标。目标1将使用荧光标记的CAS变体的活细胞成像来确定CAS粘附位点靶向的动力学和机制。CAS相互作用的蛋白质的鉴定，其本地化的重要性也将被追求。目的2将采用延时显微镜来确定CAS底物结构域磷酸化对驱动细胞运动的板状伪足突起和粘附组装/拆卸的动态过程的影响，以及对驱动入侵的podosomes-invadopodia的动态组装的影响。在相关的研究中，将研究CAS底物结构域磷酸化促进肌动蛋白聚合的能力。虽然前两个目标利用了CAS无效的小鼠成纤维细胞模型，但目标3通过使用RNA干扰来破坏CAS表达，将工作扩展到对侵袭性人乳腺癌细胞系进行建模。将评估CAS敲减对体外细胞增殖和侵袭以及体内肿瘤生长和转移的影响。用野生型与CAS突变变体的重构实验将揭示重要的功能结构域。本案无关拟议的研究将填补我们对基本信号机制理解的关键空白，该信号机制驱动对细胞运动和侵袭重要的复杂动态事件。这些事件在正常组织发育/再生和许多病理过程中具有核心作用，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新的治疗靶点和疾病的预后指标，所产生的异常运动和入侵。