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 Arp2/3 肌球蛋白 I 连接子）是从原生动物到人类表达的 1000 个残基、多结构域支架蛋白，已对其结合加帽蛋白 (CP) 并降低其与肌动蛋白丝的亲和力的能力进行了广泛研究有刺的末端。 CARMIL 蛋白似乎也在信号转导中发挥着重要作用，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 细胞表现出深刻的信号转导作用。阻断 CD28 下游的信号传导，CD28 是 T 细胞信号传导的主要共同受体（Liang et al Nat.Immunol.2013）。 在之前的工作中 (Jung et al JCB 2001)，我们表明盘基网柄菌 CARMIL 结合 CP、Arp2/3 复合物和肌球蛋白 I（通过其 SH3 结构域），并且它是肌动蛋白依赖性过程（例如趋化性和微胞饮作用）所必需的。强壮的。 在这里，我们描述了 CARMIL-GAP 的研究，CARMIL-GAP 是第二种盘基网柄菌 CARMIL，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，根据同源性，它是 GTP 酶激活 (GAP) 结构域对于 Rho-GTPa 酶。 该结构域可能具有 GAP 活性，因为全长 CARMIL-GAP 仅当该结构域包含阻止所有特征性 GAP 蛋白中的 GAP 活性的点突变 (R737A) 时才能在细胞中过表达。 与 CARMIL 一样，CARMIL-GAP 定位于富含肌动蛋白的结构，并在营养细胞和饥饿的发育细胞中表达。 一致地，通过同源重组创建的 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 CARMIL-GAP 无效细胞中表达 GFP-FL CARMIL-GAP 可以挽救吞噬作用和趋化聚集的缺陷。 使用 GAP 结构域进行的下拉的质谱分析表明，CARMIL-GAP 结合 Rac 1a，Rac 1a 是盘基网柄菌中表达的 11 种 Rac 亚型之一。 目前的工作旨在证明 CARMIL-GAP 在体内对 Rac 1a 的调​​节，并确定 CARMIL-GAP 中的哪些结构域（例如 GAP 结构域、CPI 结构域）对其功能最关键。