Genetic Analysis of FAK Activity

FAK活性的遗传分析

• 批准号: 7857976
• 负责人: David D Schlaepfer
• 金额: $ 31.51万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7857976
• 关键词: Apoptosis; Binding; Binding Sites; Biochemical; Biosensor; Blood Vessels; CD47 Antigen; Cell Adhesion; Cell Count; Cell Proliferation; Cell Survival; Cell-Matrix Junction; Cells; Complex; Defect; Development; Embryo; Event; Exhibits; Exons; Extracellular Matrix Proteins; Fibroblasts; Fibronectins; Fluorescence Resonance Energy Transfer; Focal Adhesion Kinase 1; Focal Adhesions; Genes; Guanosine Triphosphate Phosphohydrolases; Image; Immune System Diseases; In Vitro; Integrins; Investigation; Knock-in Mouse; Knock-out; Knockout Mice; Link; Malignant Epithelial Cell; Maps; Mediating; Modeling; Molecular; Monitor; Mus; N-terminal; Nuclear; Nuclear Translocation; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Point Mutation; Pregnancy; Process; Proliferating; Protein Tyrosine Kinase; Protein p53; Proteomics; Research; Role; Scaffolding Protein; Signal Transduction; Signaling Protein; Site; Staining method; Stains; TP53 gene; Talin; Testing; Time; Tumor Cell Invasion; Tyrosine Phosphorylation; Ubiquitination; Ultrasonography; Western Blotting; Work; Wound Healing; cell motility; directional cell; ezrin; genetic analysis; homologous recombination; in vivo; insight; migration; moesin; mutant; p120 GTPase Activating Protein; public health relevance; radixin protein; reconstitution; research study; rho GTPase-activating protein; tumor; tumor progression

DESCRIPTION (provided by applicant): Genetic Analysis of FAK Activity Binding interactions between extracellular matrix proteins such as fibronectin and integrins play fundamental roles during development by controlling cell adhesion, motility, and survival. Signals generated by integrins at cell attachment sites termed focal adhesions are mediated by the recruitment of cytoskeletal and signaling proteins in a manner that remain under investigation. Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is activated by integrins and hypothesized to regulate aspects of cell survival and motility during tumor progression. FAK and integrin function are essential during development as knockouts yield early embryonic lethal phenotypes. However, as FAK works as both a scaffolding protein and as a signaling kinase, knockout studies do not provide mechanistic insights in distinguishing these features of FAK action. Moreover, as FAK-null mouse embryo fibroblasts (MEFs) exhibit both proliferation and motility defects, it remains undetermined whether FAK activity is differentially involved in these events. We recently demonstrated that FAK promotes primary fibroblast proliferation through p53 inactivation in a kinase-independent manner via N-terminal FAK FERM (band 4.1, ezrin, radixin, moesin homology) domain- mediated nuclear translocation, p53 binding, and enhancement of p53 ubiquitination and turnover. Thus, we hypothesize that FAK FERM nuclear-association promotes cell survival by keeping p53 levels low. To support this model, we have generated a kinase-dead (KD) knock in point mutation (Lys-454 to Arg, R454) in exon 21 of mouse fak by homologous recombination. In the Preliminary Results, we find that homozygous KD FAK is embryonic lethal. However, unlike FAK-null MEFs that cannot grow (due to p53 activation), we find that homozygous KD FAK MEFs proliferate in culture, but show severe migration defects of enhanced focal adhesion formation and in directional motility. This shows that FAK catalytic activity is not essential for MEF proliferation-survival, but is required for cell movement in vitro and in vivo. To extend these findings, we propose 3 research aims. First, we will determine the role of FAK activity in vivo by analysis of KD FAK knock in embryos. This will involve comparisons to p53 activation in FAK-null embryos, pharmacological inhibition of FAK during development, and analysis of phospho-proteomic changes linked to FAK activity. Second, we will test whether FAK binding to and phosphorylation of talin are key events in both FAK activation and in focal adhesion turnover needed for motility. These studies will involve real-time imaging of WT, KD, and GFP-FAK reconstituted FAK-null MEFs as well as biochemical analysis of a linkage involving talin, FAK, and Src. Third, we will test the hypothesis that FAK activity promotes directionality motility-polarity via p190A RhoGAP complex formation, tyrosine phosphorylation, and selective leading-edge inhibition of RhoGTPase activity through a connection between FAK and p120RasGAP. Together, these studies will provide important insights into the molecular mechanism of cell movement underlying processes such as tumor invasion. PUBLIC HEALTH RELEVANCE: Regulated and controlled cell migration is important in development and wound healing whereas uncontrolled motility promotes immune diseases and tumor spread. Our studies are focused on understanding the molecular mechanisms governing how FAK activity differentially promotes cell motility and survival. These studies will fill key gaps in our understanding of the basic signaling events regulating cell movement and underlying processes such as tumor spread.

描述（由申请人提供）：FAK活性的遗传分析细胞外基质蛋白如纤连蛋白和整联蛋白之间的结合相互作用通过控制细胞粘附、运动和存活在发育过程中发挥重要作用。整合素在细胞粘附位点产生的信号称为粘着斑，其通过细胞骨架和信号蛋白的募集介导，其方式仍在研究中。粘着斑激酶（FAK）是一种细胞质酪氨酸激酶，被整合素激活，并被假设在肿瘤进展期间调节细胞存活和运动的方面。FAK和整联蛋白功能在发育过程中是必不可少的，因为敲除产生早期胚胎致死表型。然而，由于FAK既作为支架蛋白又作为信号激酶，敲除研究不能提供区分FAK作用的这些特征的机制见解。此外，由于FAK无效的小鼠胚胎成纤维细胞（MEFs）表现出增殖和运动缺陷，它仍然不确定是否FAK活性差异参与这些事件。我们最近证明，FAK促进原代成纤维细胞增殖，通过p53失活，在一个激酶独立的方式通过N-末端FAK FERM（带4.1，埃兹蛋白，radixin，膜突蛋白同源）结构域介导的核转位，p53结合，并增强p53泛素化和营业额。因此，我们假设FAK FERM核缔合通过保持p53水平较低来促进细胞存活。为了支持这一模型，我们已经产生了一个激酶死亡（KD）敲在点突变（Lys-454精氨酸，R454）在外显子21的小鼠fak的同源重组。在初步结果中，我们发现KD FAK纯合子是胚胎致死的。然而，与不能生长（由于p53激活）的FAK无效MEFs不同，我们发现纯合子KD FAK MEFs在培养中增殖，但显示出增强的粘着斑形成和定向运动的严重迁移缺陷。这表明FAK催化活性对于MEF增殖-存活不是必需的，但对于体外和体内的细胞运动是必需的。为了推广这些发现，我们提出了3个研究目标。首先，我们将通过分析胚胎中KD FAK敲除来确定FAK活性在体内的作用。这将涉及比较FAK无效胚胎中的p53激活，FAK在发育过程中的药理学抑制，以及与FAK活性相关的磷酸化蛋白质组学变化的分析。其次，我们将测试是否FAK结合和磷酸化的塔林是关键事件，在FAK激活和局灶性粘附周转所需的运动。这些研究将涉及WT、KD和GFP-FAK重建的FAK-null MEFs的实时成像以及涉及talin、FAK和Src的连锁的生化分析。第三，我们将测试FAK活性通过p190 A RhoGAP复合物形成、酪氨酸磷酸化和选择性前沿抑制RhoGTf 3活性（通过FAK和p120 RasGAP之间的连接）促进方向性运动极性的假设。总之，这些研究将为肿瘤侵袭等细胞运动过程的分子机制提供重要的见解。公共卫生相关性：调节和控制的细胞迁移在发育和伤口愈合中是重要的，而不受控制的运动促进免疫疾病和肿瘤扩散。我们的研究重点是了解FAK活性如何差异促进细胞运动和存活的分子机制。这些研究将填补我们对调节细胞运动的基本信号事件和肿瘤扩散等潜在过程的理解中的关键空白。