REGULATION OF ACTIN CAPPING PROTEIN

肌动蛋白加帽蛋白的调节

• 批准号: 8464156
• 负责人: JOHN A COOPER
• 金额: $ 27.87万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 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/8464156
• 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

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分解，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倒刺末端的形成是肌动蛋白的一个关键决定因素 组装和封盖这些端部是必要的，以在膜处产生力和运动。在这里， 我们研究了异源二聚体倒钩末端加帽蛋白（CP）的功能， 含有保守的CP结合基序（称为CPI）的膜相关蛋白质，但 其他方面无关。 CARMIL含有CPI基序和第二个CP结合基序，称为CSI（CARMIL-1）。 特异性相互作用），是CP的有效抑制剂，具有通过脱帽产生游离倒刺末端的能力 有帽的花丝去帽化很重要，因为细胞中CP和肌动蛋白丝的周转率 比在体外用纯化的蛋白质观察到的要快几个数量级。其他CPI基序 包括CD 2AP、Cin 85、CKIP-1、WASHCAP（FAM 21）和CapZIP的蛋白质结合CP，但抑制较少 这表明它们可能将活性CP定位于膜隔室。 目标1：加帽蛋白的调节剂如何工作？为了了解CP监管的工作原理，我们 定义了CP上的肌动蛋白结合位点，我们制作了CP与CP- 结合蛋白CPI基序与CP上的一个共同位点结合，该位点与肌动蛋白结合位点相距一定距离。 CARMIL结合引起肌动蛋白结合位点构象的变构变化。现在我们 询问各种CPI基序蛋白的细胞功能是抑制CP还是募集活性CP 到细胞中的一个位置。在体外，我们将比较蛋白质结合和抑制CP的能力， 用纯化的组分进行生物化学测定。在细胞中，我们将确定如何靶向和 CP与肌动蛋白细胞骨架的结合取决于CP相互作用蛋白及其如何与肌动蛋白相互作用。 与CP的相互作用影响局部肌动蛋白的组装和运动。 目标2：CARMIL 1在细胞中如何发挥作用？CARMIL对细胞迁移和其他肌动蛋白很重要， 多细胞动物细胞的相关功能。脊椎动物表达三种保守的CARMIL基因。我们 发现CARMIL 1和CARMIL 2对细胞迁移都很重要， 在单个迁移细胞中具有不同的非重叠功能。CARMIL 1与双GEF Trio相互作用， 激活Rac 1并刺激板状伪足形成。我们计划研究 这些表型和相互作用通过定位、生物化学、敲除和 表达方法。 约翰·库珀 1R01 GM095509-01A1