RSK-2 regulates integrin-mediated adhesion and migration

RSK-2 调节整合素介导的粘附和迁移

• 批准号: 8460455
• 负责人: Joe William Ramos
• 金额: $ 1.13万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8460455
• 关键词: Actins; Adhesions; Affect; Affinity; Binding; Biochemistry; Biological Process; Cell Adhesion; Cell Communication; Cell Survival; Cell physiology; Cell-Cell Adhesion; Cells; Complex; Confocal Microscopy; Cytoplasmic Tail; Cytoskeletal Proteins; Cytoskeleton; Data; Development; Disease; Dominant-Negative Mutation; Embryo; Extracellular Matrix; Extracellular Signal Regulated Kinases; Fibroblasts; Fibronectins; Flow Cytometry; Focal Adhesions; Genetic Transcription; Glioma; Goals; Immune Targeting; Immunofluorescence Immunologic; Inflammation; Integrins; Knockout Mice; Lead; Ligand Binding; Ligands; Link; Mediating; Modeling; Molecular; Molecular Biology; Molecular Conformation; Molecular Mechanisms of Action; Mus; Neoplasm Metastasis; Pathway interactions; Phosphorylation; Phosphotransferases; Platelet aggregation; Play; Primary Cell Cultures; Process; Protein-Serine-Threonine Kinases; Proteins; Regulation; Reporting; Research; Role; Signal Transduction; Signal Transduction Pathway; Site; Tail; Talin; Testing; Work; cancer cell; cell motility; combinatorial; filamin; in vivo; migration; mouse model; mutant; new therapeutic target; overexpression; paxillin; protein activation; ribosomal protein S6 kinase 2; scaffold; tissue/cell culture; tumor; tumor growth

Project Summary: The long-term goal of this project is to help determine the mechanisms that regulate cell adhesion and migration. Dynamic regulation of integrin affinity for ligand (activation) is important in a broad array of cellular functions including adhesion, migration, signal transduction, and the assembly of extracellular matrices. The major objective of the proposed research plan is to understand the molecular mechanism by which the ERK MAP kinase pathway modulates integrin function. RSK2 is a substrate of ERK and can mediate many of the biological functions of the Ras/ERK MAP kinase pathway including cell survival, proliferation and transcription. RSKs are large kinases consisting of two kinase domains. The regulation of RSK activity is complex and requires phosphorylation at several sites including auto-phosphorylation. We have recently shown that the protein PEA-15 binds to RSK2 and acts as a scaffold to enhance ERK activation of RSK2. Moreover, overexpression of PEA-15 can impair ERK activation of RSK2. Similarly overexpression of PEA-15 impairs ERK regulation of integrin activity and blocks cell migration. We therefore sought to determine if the effects of overexpression of PEA-15 were due to inhibition of ERK activation of RSK2. We show in the preliminary results that indeed overexpression of PEA-15 blocks RSK2 phosphorylation of filamin. Moreover dominant-active RSK2 blocks integrin activation and integrin-mediated fibronectin matrix assembly. Finally we show that the Ras/ERK affect on integrin activity is blocked by a dominant- negative RSK2 construct. Our Hypothesis is that RSK2 modulates integrin function downstream of ERK by regulating integrin ¿ tail interaction with the actin cytoskeleton. The research plan has three specific aims: (1) Establish the molecular mechanism by which RSK2 modulates integrin activation. We provide preliminary data that active RSK2 blocks integrin ligand-binding. We will investigate the molecular mechanism of this action with particular focus on the RSK2 substrate filamin and the integrin activation protein talin. (2) Determine the functional consequences of RSK2 regulation of integrin activity. Integrins are involved in cell adhesion, migration, fibronectin matrix assembly and signal transduction. We will investigate the involvement of RSK2 in these processes. (3) Investigate the mechanism of PEA-15 regulation of RSK2 activity. We have reported that PEA-15 acts as a scaffold for the activation of RSK2 by ERK. We will further determine how PEA-15 modulates RSK2 activation and how this affects cell adhesion and migration. (4) To determine how RSK2 regulation of integrin activation affects glioma invasion. We previously reported that PEA-15 expression blocks glioma invasion. We will now explore how RSK2 contributes to changes in the adhesion and invasion of glioma. These aims will be pursued in tissue culture cells using flow cytometry, confocal microscopy, immunofluorescence, molecular biology, and biochemistry. We will also employ mouse embryo fibroblasts and primary culture cells derived from mouse knock-out models. Significance: The proposed research will provide significant advances in our understanding of how the complex kinase RSK2 can play a central role in modulating cellular adhesion and migration. Changes in integrin activation are linked to diverse diseases including cancer metastasis. Many signal transduction pathways are known to modulate integrin function. However, the mechanisms by which these signals are transformed into changes in integrin conformation are not well understood. At the end of these studies we will better understand how the ERK MAP kinase signal transduction pathway modulates integrin activation through RSK2 and how this may contribute to disease states such as tumor metastasis. We will also have determined if RSK2 can serve as a potential new therapeutic target in the treatment of gliomas.

项目摘要： 该项目的长期目标是帮助确定调节细胞的机制 粘附和迁移。整联蛋白对配体的动态调节（激活）在A中很重要 广泛的蜂窝功能，包括广告，迁移，信号转导和组装 细胞外物质。拟议的研究计划的主要目标是了解 ERK MAP激酶途径调节整联蛋白功能的分子机制。 RSK2是一个 ERK的底物可以介导RAS/ERK MAP激酶的许多生物学功能 途径包括细胞存活，增殖和转录。 RSK是大型激酶，由 两个激酶结构域。 RSK活性的调节很复杂，需要在 包括自身磷酸化在内的几个站点。我们最近表明蛋白质豌豆15结合 对RSK2并充当脚手架，以增强RSK2的ERK激活。而且，过表达 PEA-15会损害RSK2的ERK激活。同样，PEA-15的过表达损害ERK 调节整联蛋白活性并阻止细胞迁移。因此，我们感觉确定效果是否 PEA-15的过表达是由于抑制RSK2的ERK激活。我们在 初步的结果确实是PEA-15的过表达阻塞丝蛋白的RSK2磷酸化。 此外，主动激活的RSK2阻断整联蛋白激活和整合素介导的纤连蛋白基质 集会。最后，我们表明RAS/ERK对整联蛋白活性的影响被主要的 - 负RSK2构建体。 我们的假设是RSK2通过调节ERK下游调节整联蛋白功能 整联蛋白»与肌动蛋白细胞骨架的尾部相互作用。研究计划具有三个具体目标： （1）建立RSK2调节整联蛋白激活的分子机制。我们提供 活跃RSK2的初步数据会阻止整联蛋白配体结合。我们将研究分子 该动作的机制特别关注RSK2底物丝蛋白和整合素 激活蛋白塔林。 （2）确定整联蛋白的RSK2调节的功能后果 活动。整合素参与细胞粘附，迁移，纤连蛋白基质组件和信号 转导。我们将研究RSK2在这些过程中的参与。 （3）调查 PEA-15 RSK2活性调节的机理。我们报告说，豌豆15充当脚手架 为了激活RSK2。我们将进一步确定PEA-15如何调节RSK2激活 以及这如何影响细胞粘合剂和迁移。 （4）确定整联蛋白的RSK2调节 激活会影响神经胶质瘤的侵袭。我们先前报道了PEA-15表达阻碍神经胶质瘤 入侵。现在，我们将探讨RSK2如何促进粘附和入侵的变化 神经胶质瘤。这些目标将在组织培养细胞中使用流式细胞术，共聚焦显微镜， 免疫荧光，分子生物学和生物化学。我们还将使用鼠标胚胎 源自小鼠敲除模型的成纤维细胞和原代培养细胞。 意义：拟议的研究将为我们理解如何 复杂的激酶RSK2可以在调节细胞粘合剂和迁移方面发挥核心作用。 整联蛋白激活的变化与包括癌症转移在内的潜水疾病有关。许多信号 已知转导途径可调节整联蛋白功能。但是， 这些信号转化为整联蛋白构象的变化，尚不清楚。在 这些研究的结尾，我们将更好地了解ERK MAP激酶信号转导途径如何 通过RSK2调节整联蛋白的激活，以及这可能有助于疾病状态 肿瘤转移。我们还将确定RSK2是否可以作为潜在的新疗法 靶向神经胶质瘤的治疗。