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.

CARMIL（加帽蛋白Arp 2/3肌球蛋白I接头）是从原生动物表达到人的1000个残基的多结构域支架蛋白，其结合加帽蛋白（CP）并降低其对肌动蛋白丝倒刺末端的亲和力的能力已被广泛研究。 CARMIL蛋白似乎也在信号转导中发挥重要作用，因为它们表现出与Rac GEF Trio的遗传和物理相互作用（Liang等人MBoC 2009; Vanderzalm等人Dev. 2009），并且缺乏CARMIL-2的T细胞在CD 28（T细胞信号传导的主要共受体）下游的信号传导中表现出深刻的阻断（Liang等Nat.Immunol.2013）。 在以前的工作中（Jung et al JCB 2001），我们发现网骨藻CARMIL结合CP、Arp 2/3复合物和肌球蛋白I（通过其SH 3结构域），并且它是肌动蛋白依赖性过程（如趋化性和微胞饮作用）所必需的。 在这里，我们描述了CARMIL-GAP的研究，第二个网骨藻CARMIL，除了所有正常的CARMIL结构域（包括CP结合CPI结构域），含有130个残基的插入，同源性，是一个GTP酶激活（GAP）结构域的Rho-GTP酶。 该结构域可能具有GAP活性，因为全长CARMIL-GAP只有在该结构域含有阻断所有特征性GAP蛋白中GAP活性的点突变（R737 A）时才能在细胞中过表达。 像CARMIL一样，CARMIL-GAP定位于富含肌动蛋白的结构，并在营养和饥饿的发育细胞中表达。 一致地，通过同源重组产生的CARMIL-GAP空细胞系在几个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，GFP-FL CARMIL-GAP在CARMIL-GAP缺失细胞中的表达挽救了吞噬作用和趋化性聚集的缺陷。 使用差距域进行的唐斯质谱分析表明，CARMIL-GAP结合Rac 1a，Rac 1a是在网骨藻中表达的11种Rac同种型之一。 目前的努力是针对证明Rac 1a在体内通过CARMIL-GAP的调节，以及鉴定CARMIL-GAP内的哪些结构域（例如差距域、CPI结构域）对其功能最关键。