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: 8144290
• 负责人: Joe William Ramos
• 金额: $ 28.22万
• 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/8144290
• 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; public health relevance; ribosomal protein S6 kinase 2; scaffold; tissue/cell culture; tumor; tumor growth

DESCRIPTION (provided by applicant): 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 2 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. PUBLIC HEALTH RELEVANCE: Control of cell adhesion is important in targeting immune cells to sites of inflammation, in platelet aggregation, and in cancer cell metastasis among other functions. We have found that the proteins PEA-15 and RSK2 may conspire to influence cell adhesion and migration. In particular we propose that PEA-15 regulates RSK2 activity while RSK2 in turn modulates the interaction of the cell cytoskeleton with adhesion proteins called integrins. At the conclusion of these studies the molecular mechanism of PEA-15 and RSK2 modulation of cell adhesion will be better understood. This will determine if these proteins are suitable targets for the development of new adhesion specific disease therapies. In particular we will test if RSK2 can serve as a potential new therapeutic target in glioma.

描述（由申请人提供）：该项目的长期目标是帮助确定调节细胞粘附和迁移的机制。整联蛋白对配体（激活）的动态调节在广泛的细胞功能中很重要，包括粘附，迁移，信号转导和细胞外矩阵组装。拟议的研究计划的主要目的是了解ERK MAP激酶途径调节整联蛋白功能的分子机制。 RSK2是ERK的底物，可以介导RAS/ERK MAP激酶途径的许多生物学功能，包括细胞存活，增殖和转录。 RSK是由两个激酶结构域组成的大型激酶。 RSK活性的调节很复杂，需要在包括自身磷酸化在内的多个部位进行磷酸化。我们最近表明，蛋白质PEA-15与RSK2结合，并充当增强RSK2 ERK激活的支架。此外，PEA-15的过表达会损害RSK2的ERK激活。同样，PEA-15的过表达会损害整联蛋白活性的ERK调节并阻止细胞迁移。因此，我们试图确定PEA-15过表达的影响是否是由于RSK2的ERK激活抑制。我们在初步结果中表明，pea-15的过度表达确实阻止了丝蛋白的RSK2磷酸化。此外，主动激活的RSK2阻断整联蛋白激活和整联蛋白介导的纤连蛋白基质组件。最后，我们表明RAS/ERK对整联蛋白活性的影响被主要的负RSK2构建体阻止。我们的假设是RSK2通过调节整合素2​​与肌动蛋白细胞骨架的相互作用来调节ERK下游的整联蛋白功能。研究计划具有三个特定的目的：（1）建立RSK2调节整联蛋白激活的分子机制。我们提供主动RSK2阻止整合素配体结合的初步数据。我们将研究这种作用的分子机制，特别关注RSK2底物丝蛋白和整联蛋白活化蛋白Talin。 （2）确定整联蛋白活性调节RSK2的功能后果。整联蛋白参与细胞粘附，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）研究PEA-15 RSK2活性调节的机理。我们报告说，PEA-15充当ERK激活RSK2的脚手架。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附和迁移。 （4）确定RSK2调节整联蛋白活化如何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的侵袭。现在，我们将探讨RSK2如何促进神经胶质瘤的粘附和侵袭。这些目标将在组织培养细胞中使用流式细胞仪，共聚焦显微镜，免疫荧光，分子生物学和生物化学。我们还将采用源自小鼠敲除模型的小鼠胚胎成纤维细胞和原代培养细胞。意义：拟议的研究将在我们对复杂激酶RSK2如何在调节细胞粘附和迁移方面发挥核心作用的理解为我们的理解提供了重大进步。整联蛋白激活的变化与包括癌症转移在内的各种疾病有关。已知许多信号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变化的机制尚不清楚。在这些研究结束时，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，以及这可能如何对肿瘤转移等疾病状态有效。我们还将确定RSK2是否可以作为治疗神经胶质瘤的潜在新治疗靶标。 公共卫生相关性：控制细胞粘附对于将免疫细胞靶向炎症部位，血小板聚集和癌细胞转移的部位以及其他功能以及其他功能很重要。我们发现，蛋白质PEA-15和RSK2可能共同影响细胞粘附和迁移。特别是我们建议PEA-15调节RSK2活性，而RSK2又调节了细胞细胞骨架与称为整联蛋白的粘附蛋白的相互作用。在这些研究结束时，将更好地理解PEA-15和RSK2调节的分子机制。这将确定这些蛋白是否是开发新粘附特定疾病疗法的合适靶标。特别是我们将测试RSK2是否可以作为神经胶质瘤的潜在新治疗靶标。