Genetic Analysis of FAK Activity

FAK活性的遗传分析

基本信息

批准号：
8272563
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
项目类别：
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/8272563
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

项目摘要

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）：通过控制细胞粘附，运动和存活，在发育过程中，细胞外基质蛋白（例如纤连蛋白和整联蛋白）之间的FAK活性结合相互作用的遗传分析起着基本作用。整联蛋白在所谓的局灶性粘连的细胞附着位点产生的信号是通过募集细胞骨架和信号蛋白的介导的，以仍在研究中的方式介导。局灶性粘附激酶（FAK）是一种细胞质酪氨酸激酶，被整联蛋白激活并假设以调节肿瘤进展过程中细胞存活和运动的各个方面。 FAK和整联蛋白功能在发育过程中至关重要，因为敲除产生早期的胚胎致死表型。但是，由于FAK既可以作为脚手架蛋白质，也可以作为信号激酶，因此敲除研究并不能在区分FAK动作的这些特征方面提供机械见解。此外，由于FAK-NULL小鼠胚胎成纤维细胞（MEF）表现出增殖和运动缺陷，因此尚不确定FAK活性是否与这些事件有差异性有关。我们最近证明，FAK通过p53通过N端FAK FERM（带4.1，Ezrin，Ezrin，radixin，Moesin同源性）结构域介导的核易位，p53结合和增强p53 Ubiquitination and Trebers的增强。因此，我们假设FAK FERM核能结合通过保持p53水平较低来促进细胞的存活。为了支持该模型，我们通过同源重组在小鼠FAK的外显子21中生成了点突变（LYS-454，r454）中的激酶死亡（KD）敲击。在初步结果中，我们发现纯合kd fak是胚胎致死的。但是，与无法生长的FAK-NULL MEF（由于p53激活）不同，我们发现纯合KD FAK MEF在培养中增殖，但显示出严重的迁移缺陷，这是增强的局灶性粘附形成和方向运动性的。这表明FAK催化活性对于MEF增殖 - 生存不是必不可少的，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3个研究目的。首先，我们将通过分析胚胎中的KD FAK敲击来确定FAK活性在体内的作用。这将涉及与FAK-NULL胚胎中p53激活的比较，在发育过程中对FAK的药理抑制以及与FAK活性相关的磷酸蛋白质变化的分析。其次，我们将测试FAK与塔林的结合和磷酸化是FAK激活和运动所需的局灶性粘附周转率的关键事件。这些研究将涉及对WT，KD和GFP-FAK的实时成像重新组成的FAK-NULL MEF，以及涉及Talin，FAK和SRC的链接的生化分析。第三，我们将检验以下假设：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之间的联系来促进方向性运动偏振度。总之，这些研究将提供有关细胞运动基础过程（例如肿瘤侵袭）的分子机制的重要见解。公共卫生相关性：受调节和受控的细胞迁移在发育和伤口愈合中很重要，而不受控制的运动性会促进免疫疾病和肿瘤扩散。我们的研究集中在理解fak活性如何差异化细胞运动和生存的分子机制上。这些研究将填补我们对调节细胞运动和诸如肿瘤扩散等基本过程的基本信号事件的理解时的关键空白。