Molecular Basis for Localization and Activation of Focal Adhesion Kinase

描述(申请人提供)：对于粘着斑激酶(FAK)如何被细胞膜上的激活剂激活，以及FAK如何调节其移位到细胞核内，我们的理解存在一个根本性的空白。FAK激活和核移位是胰腺肿瘤生长和侵袭的两个关键因素。因此，这种认识上的差距是一个重要的问题，因为它严重阻碍了有针对性的治疗干预的设计。本R03项目的目的是利用X射线结晶学揭示FAK如何通过分子内相互作用调节其核定位，以及FAK如何通过促进FAK自身磷酸化而与激活FAK的激活剂结合。这些晶体结构揭示的原子细节对于实现我的长期目标--理解和治疗控制FAK在肿瘤发生中的作用--至关重要。我的中心假设是，FAK的自磷酸化和核转位都是由位于FAK的非催化带4.1-Ezrin-Radioxin-moesin同源(FERM)结构域的核定位信号(NLS)控制的。这一假说是基于我的初步数据提出的，该数据表明FAK的焦点黏附靶向(FAT)结构域直接与该NLS结合，发表的研究表明，两个触发FAK自动磷酸化的激活剂也与该NLS结合。这项研究的基本原理是，FERM：FAT和FERM：激活子相互作用的原子细节将使我们了解FAK的自动磷酸化和核定位是如何调控的。这项R03提案的目标将通过两个具体目标实现：(1)确定分子内FERM：脂肪结构域复合体的晶体结构；(2)确定FAK FERM结构域与激活剂c-Met和PIP2之间形成的复合体的晶体结构。对于目标1，为我的初步研究建立的方案将用于生产重组FERM和FAT结构域。现有的机器人系统将用于FERM：FAT复合体的结晶形成。结构将使用基于分离的脂肪和高铁蛋白晶体结构的分子置换来确定，这些晶体结构是由我们和其他小组先前建立的。对于目标2，FERM结构域将与c-Met和PIP2的片段共结晶，这两个片段以前被证明直接与FERM结合。这项拟议的研究具有创新性，因为它将首次提供关于FAK的单一监管要素如何控制两个关键事件的机械性见解。这一贡献将是重要的，因为它将使设计同时针对FAK的自动磷酸化和核进口的多效性抑制化合物成为可能。此外，通过阻止FAK的核进口，这些抑制剂将同时阻止FAK对几个促凋亡因子(如p53和MDM2)的作用。因此，该项目将为开发针对胰腺癌最有前景的靶点的多效性PPI抑制剂的全面R01提案奠定基础。 公共卫生相关性：拟议的R03项目与公共卫生相关，因为它将有助于对FAK如何促进肿瘤存活和转移的机械性见解。针对FAK的前两种蛋白质-蛋白质相互作用(PPI)抑制剂与化疗相结合，最近被证明是非常有希望的抗胰腺癌药物。这项拟议的研究与PA的“胰腺癌试点研究”相关，因为它的结果将允许开发改进的第二代PPI抑制剂，以对抗FAK介导的胰腺肿瘤形成。

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of how focal adhesion kinase (FAK) is activated by activators at the cell membrane and how FAK regulates its translocation into the nucleus. FAK activation and nuclear translocation are two key contributors to pancreatic tumor growth and invasion. Therefore, this gap in knowledge is an important problem because it severely hampers design of targeted therapeutic intervention. The objective of this R03 project is to use x-ray crystallography to reveal how FAK regulates its nuclear localization through an intramolecular interaction and how FAK binds to activators that activate FAK by promoting FAK autophosphorylation. The atomic details revealed by these crystal structures will be essential for achieving my long-term goal of understanding and therapeutically controlling the role of FAK in tumorigenesis. My central hypothesis is that both autophosphorylation and nuclear translocation of FAK are governed by a nuclear localization signal (NLS) located on FAK's noncatalytic band4.1-ezrin-radixin-moesin homology (FERM) domain. This hypothesis is formulated on the basis of my preliminary data showing that FAK's focal adhesion targeting (FAT) domain binds directly to this NLS, and published research showing that two activators that trigger FAK autophosphorylation also bind to this NLS. The rationale for the proposed research is that the atomic details of the FERM:FAT and FERM:activator interactions will allow us to understand how autophosphorylation and nuclear localization of FAK are regulated. The objective of this R03 proposal will be achieved through two specific aims: (1) Determine the crystal structure of the intramolecular FERM:FAT domain complex; and (2) Determine the crystal structure of the complexes formed between the FAK FERM domain and the activators c-Met and PIP2. For Aim 1, protocols established for my preliminary studies will be used to produce recombinant FERM and FAT domains. Available robotics systems will be used for crystallogenesis of the FERM:FAT complex. Structures will be determined using molecular replacement based on isolated FAT and FERM crystal structures established previously by us and other groups. For Aim 2, the FERM domain will be co-crystallized with fragments from c-Met and PIP2, which were previously shown to bind to FERM directly. The proposed research is innovative because it will provide the first mechanistic insights into how a single regulatory element of FAK controls two key events. This contribution will be significant because it will enable the design of pleiotropic inhibitory compounds that simultaneously target autophosphorylation and nuclear import of FAK. Moreover, by blocking nuclear import of FAK, those inhibitors would simultaneously block FAK's action on several proapoptotic factors (such as p53 and Mdm2). Thus, this project will lay the groundwork for a full-scale R01 proposal to develop pleiotropic PPI inhibitors against a most promising target in pancreatic cancer. PUBLIC HEALTH RELEVANCE: The proposed R03 project is relevant to public health because it will contribute mechanistic insights into how FAK promotes tumor survival and metastasis. The first two protein-protein interaction (PPI) inhibitors against FAK, in combination with chemotherapy, have recently been shown to be extremely promising drugs against pancreatic cancer. The proposed research is relevant to the PA "Pilot studies in Pancreatic Cancer" because its results will allow developing improved second-generation PPI inhibitors against FAK-mediated pancreatic tumorigenesis.