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

描述（由申请方提供）：整合素连接激酶（ILK）对于贴壁依赖性细胞生长和存活、细胞周期进展、上皮细胞向间质细胞转化、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，并且在肿瘤血管生成中起重要作用。在整联蛋白的下游存在多种信号传导途径，但这些途径中的许多需要在ILK、PINCH和parvin（IPP）之间形成异源三聚体复合物。该IPP复合物在整合素-肌动蛋白和整合素-信号传导网络中充当枢纽，并且在哺乳动物系统中，IPP复合物形成先于其正确靶向粘连并且是其正确靶向粘连所需的。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。我们的目标是提高整合素信号的IPP复杂使用结构导向的功能方法的理解，并解决ILK是否具有催化活性的关键功能问题。在目标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。这些生物物理学研究将使我们能够合理地调查这种相互作用的靶向中断的细胞效应。在目标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是许多争议的主题，但据报道，IPP介导的整合素信号转导是至关重要的。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催化活性的分子基础提供结构证据。将研究潜在的底物特异性以及分子内相互作用对激酶活性的作用。使用结构引导的定点突变，我们将研究ILK催化活性在细胞中的功能作用。在目标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亚基胞质尾相互作用。如果ILK结合整联蛋白2尾，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效应。在目标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。提出的研究将提供答案的一些关键的未解决的问题，关于整合素信号的IPP复杂。它们还可以促进与癌症、心血管和炎症以及肾脏疾病的治疗相关的靶向抗ILK或抗IPP治疗剂的设计。我们的研究结果将显着提高分子，酶和功能的理解一个关键的整合素信号复合物。公共卫生相关性：该项目旨在提高我们对整合素连接激酶-PINCH- parvin（IPP）复合物的分子和功能理解。这种复合物对于胚胎发育、组织维护和修复、宿主防御和止血是必不可少的。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。拟议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。

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.