REGULATION OF ACTIN CAPPING PROTEIN

肌动蛋白加帽蛋白的调节

基本信息

批准号：
8246154
负责人：
JOHN A COOPER
金额：
$ 28.88万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-01 至 2016-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8246154
关键词：
Abbreviations Actins Address Affect Agaricales Amino Acid Motifs Binding Binding Proteins Binding Sites Biochemical Biological Assay C-terminal CD2-associated protein Cell physiology Cells Complex Cytoskeleton Defect Development Filament Genes Goals Immigration In Vitro Inflammatory Integrins Leucine-Rich Repeat Life Lipids Location Membrane Membrane Proteins Microfilaments Microscopy Modeling Molecular Molecular Conformation Movement Myosin ATPase Myosin Type I Phenotype Physiological Plus End of the Actin Filament Process Protein Binding Domain Protein Isoforms Proteins RNA Cap-Binding Proteins Recruitment Activity Regulation Research Role Shapes Site Structure Subcellular structure Surface Testing Vertebrates Work actin capping protein base cancer cell cell motility human disease inhibitor/antagonist mutant novel prevent response

Abbreviations Actins Address Affect Agaricales Amino Acid Motifs Binding Binding Proteins Binding Sites Biochemical Biological Assay C-terminal CD2-associated protein Cell physiology Cells Complex Cytoskeleton Defect Development Filament Genes Goals Immigration In Vitro Inflammatory Integrins Leucine-Rich Repeat Life Lipids Location Membrane Membrane Proteins Microfilaments Microscopy Modeling Molecular Molecular Conformation Movement Myosin ATPase Myosin Type I Phenotype Physiological Plus End of the Actin Filament Process Protein Binding Domain Protein Isoforms Proteins RNA Cap-Binding Proteins Recruitment Activity Regulation Research Role Shapes Site Structure Subcellular structure Surface Testing Vertebrates Work actin capping protein base cancer cell cell motility human disease inhibitor/antagonist mutant novel prevent response

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项目摘要

Our goals are to elucidate the molecular mechanisms that control the assembly and disassembly of actin filaments in cells and to understand how actin assembly dynamics contribute to cell movement. The creation of free barbed ends of actin filaments is a critical determinant of actin assembly, and capping those ends is necessary to produce force and movement at membranes. Here, we investigate the function of the heterodimeric barbed-end capping protein (CP) and a set of membrane-associated proteins that contain a conserved CP-binding motif, called CPI, but are otherwise unrelated. CARMIL, which contains the CPI motif and a second CP-binding motif, called CSI (CARMIL- specific interacting), is a potent inhibitor of CP with the ability to create free barbed ends by uncapping capped filaments. Uncapping is important because the turnover rates of CP and actin filaments in cells are faster by orders of magnitude than those observed with purified proteins in vitro. Other CPI-motif proteins, including CD2AP, Cin85, CKIP-1, WASHCAP(FAM21) and CapZIP bind CP but inhibit less well, suggesting that they may target active CP to membrane compartments. Aim 1: How Do Regulators of Capping Protein Work? To understand how CP regulation works, we defined the actin-binding sites on CP, and we produced crystal structures for CP in complex with CP- binding proteins. CPI motifs bind to a common site on CP at a distance from the actin-binding sites. CARMIL binding causes an allosteric change in the conformation of the actin-binding sites. Now, we ask whether the cellular function of the various CPI-motif proteins is to inhibit CP or to recruit active CP to a location in the cell. In vitro, we will compare the abilities of the proteins to bind and inhibit CP in biochemical assays with purified components. In cells, we will determine how targeting and incorporation of CP into the actin cytoskeleton depends on the CP-interacting proteins and how their interaction with CP affects local actin assembly and movement. Aim 2: How Does CARMIL1 Function in Cells? CARMIL is important for cell migration and other actin- related functions in metazoan cells. Vertebrates express three conserved CARMIL genes. We discovered that CARMIL1 and CARMIL2 are both important for cell migration and that they have distinct non-overlapping functions in a single migrating cell. CARMIL1 interacts with the dual-GEF Trio, activates Rac1, and stimulates lamellipodia formation. We plan to investigate the molecular basis of these phenotypes and interactions by a combination of localization, biochemical, knockdown, and expression approaches. COOPER, John A. 1R01 GM095509-01A1

我们的目标是阐明控制组装的分子机制细胞中肌动蛋白丝的拆卸，并了解肌动蛋白组装动力学如何有助于细胞运动。肌动蛋白丝的自由刺末端的创建是肌动蛋白的关键决定因素组装和封顶对于在膜上产生力和运动是必要的。这里，我们研究了异二聚体栏杆末端上限蛋白（CP）的功能和一组膜相关蛋白包含保守的CP结合基序，称为CPI，但是否则无关。卡米尔包含CPI基序和第二个CP结合基序，称为CSI（Carmil- 特定的相互作用），是CP的有效抑制剂，能够通过解衬盖创建自由刺端的末端封顶的细丝。拆卸很重要，因为CP和肌动蛋白细胞的周转率比在体外观察到的纯化蛋白观察到的数量级要快。其他CPI-MOTIF 蛋白质，包括CD2AP，CIN85，CKIP-1，WASHCAP（FAM21）和CAPZIP结合CP，但抑制较少好吧，这表明它们可以将活动CP靶向膜室。目标1：封盖蛋白质的调节剂如何工作？要了解CP监管的工作原理，我们定义了CP上的肌动蛋白结合位点，我们与CP-cp-Cp-cp-生产了CP的晶体结构结合蛋白。 CPI基序与CP上的公共位点结合，距离肌动蛋白结合位点的距离。卡米尔结合会导致肌动蛋白结合位点构象的变构变化。现在，我们询问各种CPI-MOTIF蛋白的细胞功能是抑制CP还是募集活动CP 到单元中的位置。在体外，我们将比较蛋白质结合和抑制CP的能力具有纯化成分的生化测定。在细胞中，我们将确定目标和将CP掺入肌动蛋白细胞骨架中取决于CP相互作用的蛋白质及其如何与CP的相互作用会影响局部肌动蛋白组装和运动。 AIM 2：Carmil1如何在细胞中起作用？卡米尔对于细胞迁移和其他肌动蛋白很重要 - 相关功能在后生细胞中。脊椎动物表达三个保守的卡米尔基因。我们发现Carmil1和Carmil2对细胞迁移都很重要，并且它们具有单个迁移单元中不同的非重叠函数。 Carmil1与双GEF三重奏相互作用，激活Rac1并刺激片状脂蛋白形成。我们计划研究这些表型和相互作用通过本地化，生化，敲低和表达方法。库珀，约翰·A。 1R01 GM095509-01A1