Molecular basis of ILK-mediated cell adhesion

ILK介导的细胞粘附的分子基础

• 批准号: 8513731
• 负责人: JUN QIN
• 金额: $ 37.72万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8513731
• 关键词: Actins; Active Sites; Affect; Amino Acid Sequence; Ankyrin Repeat; Binding; Biochemical; C-terminal; Cell Adhesion; Cell Shape; Cell physiology; Cell-Cell Adhesion; Cells; Clinical; Communication; Complex; Coupled; Couples; Cytoplasmic Tail; Cytoskeleton; Data; Disease; Eph Family Receptors; Ephrins; Event; Extracellular Matrix; Extracellular Matrix Proteins; F-Actin; Figs - dietary; Focal Adhesions; Funding; G Actin; Genetic; Grant; Heart Neoplasms; Heart failure; Integrins; Investigation; LIMS1 gene; Lead; Life; Link; Mediating; Molecular; Mutation; N-terminal; Pathway interactions; Peptide Sequence Determination; Phosphotransferases; Play; Process; Protein-Serine-Threonine Kinases; Proteins; Receptor Signaling; Regulation; Reporting; Research; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site; Stress Fibers; Testing; Time; United States National Institutes of Health; adhesion process; base; cell motility; design; interest; migration; novel; protein complex; public health relevance; receptor; response; scaffold; therapeutic development

DESCRIPTION (provided by applicant): This competitive renewal requests continued support for our investigation on ILK, a central component of supramolecular integrin adhesome involved in regulating diverse cellular processes such as cell shape change, migration, and survival. Discovered in 1996, ILK has long been thought to function as a serine- threonine kinase that phosphorylates cytoplasmic tails (CT) of integrin receptors and transmit signals between ECM and actin cytoskeleton. Highly elevated in failing hearts and tumors, ILK has also been pursued as a "hot" kinase target for therapeutics development. However, our recent structural analysis revealed that ILK contains a severely degraded active site. Extensive biochemical analyses, in parallel with genetic data, corroborated this observation, strongly indicating that ILK is a pseudokinase. Our finding thus challenges a 16-year dogma, signifying a major new direction of the ILK research as indicated by a dozen recent review articles. Our immediate questions are: (i) without catalytic function, how does ILK mediate the integrin-actin communication and other signaling events? (ii) Given the clinical importance of ILK, what might be a new strategy for the ILK- based therapy? In preliminary studies, we found that ILK utilizes two distinct interfaces, one at its N-terminal ankyrin repeat domain (ARD) and the other at the pseudoactive site of the C-terminal kinase-like domain (KLD), to bind adaptors PINCH and Parvin respectively, leading to the formation of a tight heterotrimer (IPP). We further discovered that this ILK-centered heterotrimer binds G-actin and promotes dynamic F-actin assembly. These findings provide exciting clues for unraveling the mechanism of ILK function. In particular, they potentially explai a long-standing puzzle why loss of ILK drastically impairs the actin cytoskeleton and inhibits cell migration. Since ILK also binds to integrin and other signaling molecules, we are entering into a new era for elucidating how ILK may act as a central scaffolding machinery to regulate cytoskeleton remodeling and cell adhesion. In the next funding period, we will rigorously examine this machinery by determining the detailed structural basis of ILK-mediated actin assembly via IPP (Aim1) and elucidate how this actin assembly process is coupled with ECM via a novel integrin-IPP-actin pathway (Aim2). We will further investigate how this pathway may cross-talk with other pathways for cooperative regulation of cell adhesion dynamics (Aim3). Overall, our proposal represents a major programmatic shift for the ILK research. The results will provide a new and comprehensive view of the ILK-mediated signaling and cell adhesion. They may also impact on our understanding of multiple ILK-associated diseases and also re-invigorate strategies for the ILK-targeted therapy.

描述（由申请人提供）：这种竞争性更新请求继续支持我们对ILK的调查，这是涉及调节各种细胞过程（例如细胞形状变化，迁移和生存）的超分子整合素的核心组成部分。在1996年发现，长期以来，ILK一直被认为是丝氨酸 - 苏氨酸激酶，可磷酸化整联蛋白受体的细胞质尾巴（CT），并在ECM和肌动蛋白细胞骨架之间发射信号。在心脏和肿瘤失败的情况下，ILK也被追求是治疗剂开发的“热”激酶靶标。但是，我们最近的结构分析表明，ILK包含一个严重退化的活性位点。广泛的生化分析与遗传数据并行，证实了这一观察结果，强烈表明幼稚是一种假激酶。因此，我们的发现挑战了一项16年的教条，这表明了《幼儿园研究》的主要新方向，如最近的十几篇评论文章所示。我们的直接问题是：（i）如果没有催化功能，ILK如何介导整联蛋白 - 肌动蛋白的通信和其他信号事件？ （ii）鉴于ILK的临床重要性，基于ILK的治疗的新策略可能是什么？在初步研究中，我们发现ILK利用了两个不同的接口，一个 在其N末端氨基链蛋白重复结构域（ARD）和另一个在C-末端激酶样结构域（KLD）的伪活性位点上，分别结合适配器捏合体和Parvin，导致形成紧密的异核体（IPP）。我们进一步发现，这种以ILK为中心的异构体结合G-肌动蛋白并促进动态F-肌动蛋白组装。这些发现为揭示了ILK功能的机制提供了令人兴奋的线索。特别是，它们可能会激发出一个长期的难题，为什么伊尔克的流失会大大损害肌动蛋白细胞骨架并抑制细胞 迁移。由于ILK也与整联蛋白和其他信号分子结合，因此我们进入了一个新时代，以阐明ILK如何充当中央脚手架机械，以调节细胞骨架重塑和细胞粘附。在下一个资金期间，我们将通过IPP（AIM1）确定ILK介导的肌动蛋白组装的详细结构基础，并阐明该肌动蛋白组装过程如何通过新型的整合素-IPP-actin途径与ECM结合（AIM2）。我们将进一步研究该途径如何与其他途径交叉，以调节细胞粘附动力学（AIM3）。总体而言，我们的建议代表了ILK研究的重大程序转变。结果将为ILK介导的信号传导和细胞粘附提供新的全面视图。它们还可能影响我们对多种ILK相关疾病的理解，并重新为靶向ILK靶向治疗的策略。