Control of actin assembly in cells through regulation of Capping Protein

通过调节加帽蛋白来控制细胞中肌动蛋白的组装

• 批准号: 9787942
• 负责人: JOHN A HAMMER
• 金额: $ 75.82万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9787942
• 关键词: Actins; Affinity; Animals; Binding; Biochemical; CD28 gene; Cell Line; Cell physiology; Cells; Chemotaxis; Complex; Defect; Dictyostelium; Exhibits; GTPase-Activating Proteins; Gene Targeting; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; Image; Length; Mus; Myosin Type I; Null Lymphocytes; Phagocytosis; Play; Plus End of the Actin Filament; Point Mutation; Process; Property; Protein Isoforms; Proteins; Protozoa; RNA Cap-Binding Proteins; Regulation; Role; SH3 Domains; Scaffolding Protein; Signal Transduction; Structure; T-Cell Receptor; T-Lymphocyte; Work; base; cell motility; homologous recombination; in vivo; man; mutant; myotrophin; overexpression; protein purification; rho GTP-Binding Proteins; structural biology

CARMILs (Capping protein Arp2/3 Myosin I Linker) are 1000 residue, multi-domain scaffold proteins expressed from protozoa to man that have been studied extensively with regard to their ability to bind Capping Protein (CP) and reduce its affinity for the actin filament barbed end. CARMIL proteins also appear to play important roles in signal transduction, as they exhibit genetic and physical interactions with the Rac GEF Trio (Liang et al MBoC 2009; Vanderzalm et al. Dev. 2009), and T cells lacking CARMIL-2 exhibit a profound block in signaling downstream of CD28, the major co-receptor for T cell signaling (Liang et al Nat. Immunol. 2013). In previous work (Jung et al JCB 2001), we showed that Dictyostelium CARMIL binds CP, the Arp2/3 complex and myosin I (through its SH3 domain), and it is required for actin-dependent processes such as chemotaxis and micropinocytosis to be robust. Here we describe studies of CARMIL-GAP, a second Dictyostelium CARMIL that contains, in addition to all the normal CARMIL domains (including the CP binding CPI domain), a 130 residue insertion that, by homology, is a GTPase activating (GAP) domain for Rho-GTPases. This domain probably possesses GAP activity given that full length CARMIL-GAP can only be over-expressed in cells if this domain contains a point mutation (R737A) that blocks GAP activity in all characterized GAP proteins. Like CARMIL, CARMIL-GAP localizes to actin-rich structures and is expressed in both vegetative and starved, developing cells. Consistently, CARMIL-GAP null cell lines created by homologous recombination exhibit pronounced defects in several actin-based processes, including phagocytosis, motility and chemotactic aggregation. Importantly, expression of GFP-FL CARMIL-GAP in CARMIL-GAP null cells rescues the defects in phagocytosis and chemotactic aggregation. Mass spec analyses of pull downs made using the GAP domain indicate that CARMIL-GAP binds Rac 1a, one of eleven Rac isoforms expressed in Dictyostelium. Current efforts are directed at demonstrating regulation of Rac 1a by CARMIL-GAP in vivo, and at identifying which domains within CARMIL-GAP (e.g. the GAP domain, the CPI domain) are most critical for its function.

Carmils（封盖蛋白ARP2/3肌球蛋白I连接器）是1000个残基，多域支架蛋白，这些蛋白质从原生动物表达到人类的人，这些蛋白已广泛研究了其结合蛋白（CP）的能力，并降低了其对肌动蛋白丝线丝带末端的亲和力。 卡米尔蛋白在信号转导中似乎也起着重要作用，因为它们表现出与RAC GEF三人组的遗传和物理相互作用（Liang等人MBOC 2009； Vanderzalm等人Dev。2009），而缺乏Carmil-2的T细胞在CD28的信号传导下表现出深刻的障碍。 在先前的工作（Jung等人JCB 2001）中，我们表明carmil结合了CP，ARP2/3复合物和肌球蛋白I（通过其SH3结构域），对于肌动蛋白依赖性过程（例如化学恰好和微骨细胞增多）是必需的。 在这里，我们描述了Carmil-GAP的研究，Carmil-GAP是第二个Carmil的Carmil-GAP，除了所有正常的Carmil结构域（包括CP结合CPI结构域）之外，还包含130个残基插入，它是同源性的130个残基插入，是Rho-GTPases的GTPase激活（GAP）域。 鉴于该域中包含一个点突变（R737A），该域只能在细胞中过度表达CARMIL间隙（R737A），该域才能在所有表征的GAP蛋白中阻断GAP活性，因此该域可能具有GAP活性。 像卡米尔一样，卡米尔 - gap植物本地化为富含肌动蛋白的结构，并在营养和饥饿的开发细胞中表达。 一致地，由同源重组产生的Carmil-GAP无效细胞系在几种基于肌动蛋白的过程中表现出明显的缺陷，包括吞噬作用，运动性和趋化性聚集。 重要的是，在卡米尔间隙无效细胞中GFP-FL CARMIL-GAP的表达挽救了吞噬作用和趋化性聚集的缺陷。 使用间隙结构域进行的下拉倾角的质量规格分析表明，卡米尔间隙结合了RAC 1A，RAC 1A，这是用dictyostelium表达的11种RAC同工型之一。 当前的努力是通过体内Carmil-gap来证明RAC 1A的调节，并确定Carmil-GAP中哪些域（例如GAP域，CPI域）对其功能最为重要。