Genetic Analysis of FAK Activity

描述(申请人提供)：FAK活性的遗传分析纤维连接蛋白和整合素等细胞外基质蛋白之间的结合相互作用通过控制细胞黏附、运动和生存在发育过程中发挥基本作用。整合素在细胞附着部位产生的信号称为局部粘连，是通过细胞骨架和信号蛋白的募集来调节的，这种方式仍在研究中。粘着斑激酶(FAK)是一种细胞质酪氨酸激酶，由整合素激活，在肿瘤进展过程中调节细胞存活和运动的各个方面。FAK和整合素功能在发育过程中是必不可少的，因为敲除会产生早期胚胎致命表型。然而，由于FAK既是一种支架蛋白，也是一种信号转导蛋白，基因敲除研究并不能提供区分FAK作用的这些特征的机械性见解。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，目前还不确定FAK活性是否与这些事件有关。我们最近证实，FAK通过N端FAK FERM(Band 4.1，Ezrin，Radisin，Moesin Homology)结构域介导的核转位，P53结合，以及促进P53泛素化和周转，以一种不依赖于蛋白激酶的方式，促进原代成纤维细胞的增殖。因此，我们假设FAK FERM核关联通过保持较低的P53水平来促进细胞存活。为了支持这一模型，我们通过同源重组在小鼠FAK的第21外显子上产生了一个Kd敲入点突变(Lys-454到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缺失的MEF不能生长(由于P53激活)不同，我们发现纯合子的KD FAK MEF在培养中可以增殖，但表现出严重的迁移缺陷，即增强的局部黏附形成和定向运动。这表明FAK的催化活性不是MEF增殖和存活所必需的，而是细胞在体外和体内运动所必需的。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KD FAK在胚胎中的敲打来确定FAK活性在体内的作用。这将涉及到与FAK缺失胚胎中P53激活的比较，FAK在发育过程中的药物抑制，以及与FAK活性相关的磷酸化蛋白质组变化的分析。其次，我们将测试FAK与Talin的结合和磷酸化是否是FAK激活和运动所需的焦点黏附转换的关键事件。这些研究将涉及WT、KD和GFP-FAK重组FAK缺失的MEF的实时成像，以及涉及talin、FAK和Src的连锁的生化分析。第三，我们将验证这样的假设，即FAK活性通过p190A RhoGAP复合体的形成、酪氨酸磷酸化以及通过FAK和p120RasGAP之间的联系选择性地抑制RhoGTPase活性来促进方向性运动-极性。总之，这些研究将为肿瘤侵袭等过程中细胞运动的分子机制提供重要的见解。公共卫生相关性：有控制的细胞迁移在发育和伤口愈合中很重要，而不受控制的运动会促进免疫性疾病和肿瘤的扩散。我们的研究集中在了解FAK活性如何不同地促进细胞运动和生存的分子机制。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。

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.