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

描述(申请人提供)：整合素连接的激酶(ILK)对锚定依赖的细胞生长和存活、细胞周期进展、上皮细胞到间充质细胞的转变、细胞运动、收缩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，并在肿瘤血管生成中发挥重要作用。整合素下游有多条信号通路，但其中许多通路需要在ILK、Pinch和Parvin(IPP)之间形成一个异三聚体复合体。这种IPP复合体在整合素-肌动蛋白和整合素信号网络中起着枢纽的作用，在哺乳动物系统中，IPP复合体的形成是其正确靶向粘连所必需的。目前，我们对IPP复合体如何形成，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IPP复合体对整合素信号的理解，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，以提供对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理性地研究这种相互作用的定向中断对细胞的影响。在目标2中，我们将提供ILK激活域的结构、酶和功能分析。这种假激酶的催化能力仍然是争议很大的主题，但据报道在IPP介导的整合素信号转导中起着关键作用。我们将解开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。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亚单位胞浆尾巴相互作用。如果ILK与整合素2尾结合，我们将确定这种相互作用的结构基础，并研究其靶向干扰的功能影响。在目标4中，我们将确定完整的IPP络合物的分子结构。这一长期目标将提供IPP复合体如何形成的结构性描述，并将允许对其细胞角色进行有针对性的功能分析。所提出的研究将为关于IPP复合体整合素信号转导的一些关键悬而未决的问题提供答案。它们还可以促进与癌症、心血管疾病、炎症性疾病和肾脏疾病的治疗相关的靶向抗ILK或抗IPP疗法的设计。我们的结果将显著提高对一个关键的整合素信号复合体的分子、酶和功能的理解。与公众健康相关：该项目旨在加强我们对整合素连接的激酶-夹杂-帕尔文(IPP)复合体的分子和功能的了解。这种复合体对胚胎发育、组织维护和修复、宿主防御和止血都是必不可少的。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复合体如何调节其关键的细胞角色。

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.