Genetic Analysis of FAK Activity

FAK活性的遗传分析

• 批准号: 8074495
• 负责人: David D Schlaepfer
• 金额: $ 31.19万
• 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/8074495
• 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; Cytoskeletal Proteins; 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; Health; 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; 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; radixin protein; reconstitution; research study; 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.

细胞外基质蛋白（如纤维连接蛋白和整合素）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起着重要作用。整合素在称为局灶黏附的细胞附着位点产生的信号是由细胞骨架蛋白和信号蛋白的募集介导的，其方式仍在研究中。局灶黏附激酶（FAK）是一种细胞质酪氨酸激酶，被整合素激活，并被认为在肿瘤进展过程中调节细胞存活和运动。FAK和整合素功能在发育过程中至关重要，因为敲除会产生早期胚胎致死表型。然而，由于FAK同时作为支架蛋白和信号激酶，敲除研究并不能提供区分FAK作用这些特征的机制见解。此外，由于FAK缺失的小鼠胚胎成纤维细胞（mef）同时表现出增殖和运动缺陷，因此尚不确定FAK活性是否在这些事件中有差异参与。我们最近证明，FAK通过n端FAK FERM （band 4.1, ezrin, radixin， moesin同源）结构域介导的核易位、p53结合以及p53泛素化和周转增强，以激酶不依赖的方式通过p53失活促进原代成纤维细胞增殖。因此，我们假设FAK - FERM核关联通过保持p53水平较低来促进细胞存活。为了支持这一模型，我们通过同源重组在小鼠fak的21外显子上产生了一个激酶死亡（KD）敲入点突变（Lys-454 To Arg, R454）。在初步结果中，我们发现纯合子KD - FAK具有胚胎致死性。然而，与FAK-null mef不能生长（由于p53激活）不同，我们发现纯合子KD - FAK mef在培养中增殖，但表现出严重的迁移缺陷，增强了局灶粘连形成和定向运动性。这表明FAK的催化活性不是MEF增殖存活所必需的，而是体外和体内细胞运动所必需的。为了扩展这些发现，我们提出了三个研究目标。首先，我们将通过分析胚胎中的KD - FAK敲除来确定FAK活性在体内的作用。这将包括与无FAK胚胎中p53激活的比较，发育过程中FAK的药理抑制，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FAK与talin的结合和磷酸化是否是FAK激活和运动所需的局灶黏附转换的关键事件。这些研究将包括WT、KD和GFP-FAK重建的FAK-null mef的实时成像，以及talin、FAK和Src连锁的生化分析。第三，我们将验证FAK活性通过p190A RhoGAP复合物形成、酪氨酸磷酸化以及通过FAK和p120RasGAP之间的联系选择性抑制RhoGTPase活性来促进方向性、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：调节和控制细胞迁移在发育和伤口愈合中很重要，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活性如何以不同的方式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和潜在过程（如肿瘤扩散）的基本信号事件的理解的关键空白。