Molecular Basis for Localization and Activation of Focal Adhesion Kinase

粘着斑激酶定位和激活的分子基础

• 批准号: 8358453
• 负责人: Stefan T Arold
• 金额: $ 7.9万
• 依托单位: UNIVERSITY OF TX MD ANDERSON CAN CTR
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-10 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8358453
• 关键词: Adenosine Triphosphate; Adult; Apoptotic; Binding; Cell Nucleus; Cell Survival; Cell membrane; Cell surface; Cells; Complex; Crystallography; Data; Development; Event; Focal Adhesion Kinase 1; Focal Adhesions; Gene Targeting; Generations; Goals; Growth; Hot Spot; Immunotherapy; Knowledge; Ligands; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of pancreas; Mediating; Modeling; Molecular; Molecular Target; Mus; Mutation; Neoplasm Metastasis; Nuclear; Nuclear Import; Nuclear Localization Signal; Nuclear Translocation; Outcome; Pharmaceutical Preparations; Phosphatidylinositol 4,5-Diphosphate; Phospholipids; Phosphotransferases; Pilot Projects; Protocols documentation; Public Health; Publishing; Recombinants; Regulatory Element; Research; Robotics; Role; Signal Transduction; Structure; System; Testing; Therapeutic Intervention; Tissues; Tumor Cell Invasion; Work; analog; base; cancer cell; cancer recurrence; chemotherapy; combat; design; dosage; drug development; ezrin; improved; inhibitor/antagonist; innovation; insight; meetings; moesin; neoplastic cell; overexpression; pancreatic neoplasm; pancreatic tumorigenesis; preclinical study; protein protein interaction; radixin protein; tumor; tumor growth; tumorigenesis

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.

描述（由申请人提供）：我们对焦点粘附激酶（FAK）如何被细胞膜上的激活剂激活以及FAK如何调节其转移到核中的基本差距。 FAK激活和核易位是胰腺肿瘤生长和侵袭的两个关键因素。因此，这种知识的差距是一个重要的问题，因为它严重阻碍了目标治疗干预的设计。该R03项目的目的是使用X射线晶体学来揭示FAK如何通过分子内相互作用来调节其核定位，以及FAK如何与激活剂结合通过促进FAK自磷酸化来激活FAK的激活剂。这些晶体结构所揭示的原子细节对于实现我理解和治疗控制FAK在肿瘤发生中的作用的长期目标至关重要。我的中心假设是，FAK的自磷酸化和核易位均由位于FAK的非催化频段4.1-柔蛋白 - 雷迪克西蛋白 - 摩西蛋白（FERM）域上的核定位信号（NLS）控制。该假设是根据我的初步数据提出的，表明FAK的局灶性靶向（FAT）结构域直接与该NLS结合，并发表的研究表明，两个触发FAK自磷酸化的激活因子也与该NLS结合。拟议的研究的理由是，FERM的原子细节：脂肪和FERM：激活剂相互作用将使我们能够了解FAK的自磷酸化和核定位。该R03提案的目的将通过两个具体的目的来实现：（1）确定分子内FERM的晶体结构：脂肪结构域复合物； （2）确定FERM域和激活剂C-MET和PIP2之间形成的复合物的晶体结构。对于AIM 1，为我的初步研究建立的方案将用于生产重组FERM和脂肪领域。可用的机器人系统将用于FERM：脂肪复合物的结晶。将使用基于我们和其他组先前建立的分离脂肪和FERM晶体结构的分子替代品确定结构。对于AIM 2，FERM结构域将与C-Met和pip2的片段共结晶，这些片段先前被证明直接与FERM结合。拟议的研究具有创新性，因为它将为FAK的单个调节元素如何控制两个关键事件提供第一个机械见解。这项贡献将是重要的，因为它将能够设计多效性抑制性化合物，这些化合物同时以FAK的自磷酸化和核进口为目标。此外，通过阻止FAK的核进口，这些抑制剂将同时阻止FAK对几个促凋亡因素（例如p53和MDM2）的行动。因此，该项目将为全面的R01提案奠定基础，以开发对胰腺癌中最有希望的靶标的多效性PPI抑制剂。 公共卫生相关性：拟议的R03项目与公共卫生有关，因为它将为FAK如何促进肿瘤生存和转移提供机械洞察。最近，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，最近已被证明是针对胰腺癌的极有前途的药物。拟议的研究与PA的“胰腺癌试验研究”有关，因为它的结果将允许改善针对FAK介导的胰腺肿瘤发生的第二代PPI抑制剂。