Structure and function of the integrin-linked kinase-PINCH-parvin complex

整合素连接激酶-PINCH-parvin复合物的结构和功能

• 批准号: 8113966
• 负责人: Titus Jonathon Boggon
• 金额: $ 34.06万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-17 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8113966
• 关键词: Actins; Adaptor Signaling Protein; Adhesions; Affinity; Ankyrin Repeat; Architecture; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Blood Platelets; Blood Vessels; Brain; C-terminal; Cardiac; Cardiovascular system; Cell Adhesion; Cell Cycle Progression; Cell Survival; Cell physiology; Cells; Chondrocytes; Competence; Complex; Cytoplasmic Protein; Cytoplasmic Tail; Data; Development; Embryonic Development; Fishes; Foundations; Goals; Growth Factor; Health; Hemostatic function; Host Defense; In Vitro; Inflammatory; Integrin Binding; Integrins; Interruption; Kidney; Kidney Diseases; LIM Domain; LIMS1 gene; Maintenance; Measures; Mediating; Molecular; Mus; Muscle; Mutagenesis; Mutation; N-terminal; Pathway interactions; Phosphotransferases; Play; Protein Kinase; Proteins; Protocols documentation; Reporting; Resolution; Roentgen Rays; Role; Sequence Analysis; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Skin; Structure; Substrate Specificity; System; T-Lymphocyte; Tail; Testing; Therapeutic; Tissues; Tumor Angiogenesis; X-Ray Crystallography; base; cancer therapy; cell growth; cell motility; design; epithelial to mesenchymal transition; fly; genetic analysis; high throughput screening; improved; integrin-linked kinase; intermolecular interaction; migration; repaired; tumor

DESCRIPTION (provided by applicant): The integrin-linked kinase (ILK) is critical for anchorage-dependent cell growth and survival, cell cycle progression, epithelial to mesenchymal transition, cell motility, contractility and early development. ILK is also required for cardiac, vascular, brain, kidney, muscle, skin, platelet, chondrocyte and T cell function and plays important roles in tumor angiogenesis. There are multiple signaling pathways downstream of integrins but many of these pathways require the formation of a heterotrimeric complex between ILK, PINCH and parvin (IPP). This IPP complex serves as a hub in integrin-actin and integrin-signaling networks, and in mammalian systems IPP complex formation precedes and is required for its correct targeting to adhesions. There are currently significant deficiencies in our understanding of how the IPP complex forms, how it interacts with integrins, and whether it is enzymatically competent. We aim to improve the understanding of integrin signaling by the IPP complex using a structure-directed functional approach and to resolve the key functional question of whether ILK is catalytically active. In Aim 1 we will determine crystal structures and conduct binding studies to provide a comprehensive molecular description of the interaction between ILK and PINCH. These biophysical studies will allow us to rationally investigate the cellular effects of targeted interruption of this interaction. In Aim 2 we will provide structural, enzymatic and functional analyses of the ILK kinase domain. The catalytic competence of this pseudokinase remains the subject of much controversy, but is reported to be critical for IPP-mediated integrin signaling. We will solve crystal structures of the ILK kinase domain and assess its catalytic activity to provide structural evidence for the molecular basis of ILK catalytic competence. Potential substrate specificities will be studied as will the role of intramolecular interactions on kinase activity. Using structure-guided site-directed mutagenesis we will investigate the functional role of ILK catalytic activity in cells. In Aim 3 we will determine the molecular basis for ILK interactions with parvin and whether ILK directly interacts with integrin 2 subunit cytoplasmic tails. If ILK binds integrin 2 tails we will determine the structural basis for this interaction and investigate the functional effects of its targeted disruption. In Aim 4 we will determine the molecular architecture of the complete IPP complex. This long-term goal will provide a structural description of how the IPP complex forms and will allow targeted functional analysis of its cellular role. The studies proposed will provide answers to some of the critical unresolved questions regarding integrin signaling by the IPP complex. They may also facilitate the design of targeted anti-ILK or anti-IPP therapeutics relevant to the treatment of cancer, cardiovascular and inflammatory and kidney diseases. Our results will significantly enhance the molecular, enzymatic and functional understanding of a critical integrin signaling complex. PUBLIC HEALTH RELEVANCE: The project aims to enhance our molecular and functional understanding of integrin-linked kinase-PINCH- parvin (IPP) complex. This complex is essential for embryonic development, tissue maintenance and repair, host defense and hemostasis. We will determine crystal structures, conduct functional studies and describe the effects of mutations in cells. The proposed studies will help us understand how the IPP complex mediates its critical cellular roles.

描述（由申请人提供）：整合素连接激酶（ILK）对锚定依赖性细胞生长和存活、细胞周期进展、上皮向间质转化、细胞运动性、收缩性和早期发育至关重要。ILK也是心脏、血管、脑、肾、肌肉、皮肤、血小板、软骨细胞和T细胞功能所必需的，在肿瘤血管生成中起重要作用。整合素的下游有多种信号通路，但其中许多通路需要在ILK、PINCH和parvin （IPP）之间形成异三聚体复合物。这种IPP复合物是整合素-肌动蛋白和整合素信号网络的枢纽，在哺乳动物系统中，IPP复合物的形成先于其正确靶向粘附。目前，我们对IPP复合物如何形成、它如何与整合素相互作用以及它是否具有酶活性的理解存在重大缺陷。我们的目标是利用结构导向的功能方法提高对IPP复合体整合素信号传导的理解，并解决ILK是否具有催化活性的关键功能问题。在Aim 1中，我们将确定晶体结构并进行结合研究，以提供ILK和PINCH之间相互作用的全面分子描述。这些生物物理研究将使我们能够合理地研究有针对性地中断这种相互作用的细胞效应。在Aim 2中，我们将提供ILK激酶结构域的结构、酶和功能分析。这种假激酶的催化能力仍然存在很多争议，但据报道，它对ipp介导的整合素信号传导至关重要。我们将解析ILK激酶结构域的晶体结构并评估其催化活性，为ILK催化能力的分子基础提供结构证据。潜在的底物特异性将被研究，以及分子内相互作用对激酶活性的作用。利用结构导向的位点定向诱变，我们将研究ILK在细胞中催化活性的功能作用。在Aim 3中，我们将确定ILK与parvin相互作用的分子基础，以及ILK是否直接与整合素2亚基细胞质尾部相互作用。如果ILK结合整合素2尾部，我们将确定这种相互作用的结构基础，并研究其靶向破坏的功能影响。在Aim 4中，我们将确定完整IPP复合物的分子结构。这一长期目标将提供IPP复合物如何形成的结构描述，并将允许对其细胞作用进行有针对性的功能分析。提出的研究将为一些关于整合素信号通过IPP复合体的关键未解决的问题提供答案。它们还可能有助于设计与治疗癌症、心血管疾病、炎症和肾脏疾病相关的靶向抗ilk或抗ipp疗法。我们的研究结果将显著增强对一个关键整合素信号复合物的分子、酶和功能的理解。公共卫生相关性：该项目旨在增强我们对整合素连接激酶- pinch - parvin （IPP）复合物的分子和功能的理解。这种复合物对胚胎发育、组织维护和修复、宿主防御和止血至关重要。我们将确定晶体结构，进行功能研究并描述细胞突变的影响。提出的研究将帮助我们了解IPP复合物如何介导其关键的细胞作用。