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(Capping Protein Arp2/3 myosin I Linker)是原生动物表达的1000个残基、多结构域的支架蛋白，其结合覆盖蛋白(CP)和降低其与肌动蛋白细丝带刺末端的亲和力已被广泛研究。CARMIL蛋白似乎也在信号转导中发挥重要作用，因为它们与RAC环境基金三重奏表现出遗传和物理上的相互作用(梁等人，MBoC，2009；Vanderzalm等人。戴夫。2009年)，缺乏CARMIL-2的T细胞在CD28下游的信号传递方面表现出严重的阻断，CD28是T细胞信号的主要共同受体(梁等人NAT)。免疫系统。2013年)。在之前的工作(Jung Et Al JCB 2001)中，我们发现DictyostelialCARMIL通过其SH3结构域与CP、Arp2/3复合体和肌球蛋白I结合，并且它是肌动蛋白依赖过程所必需的，如趋化和微胞吞噬。在这里，我们描述了对CARMIL-GAP的研究，这是第二个CARMIL，除了所有正常的CARMIL结构域(包括CP结合的CPI结构域)外，还包含一个130个残基的插入，根据同源性，它是Rho-GTP酶的GTP酶激活(GAP)结构域。该结构域可能具有GAP活性，因为只有当该结构域包含阻止所有特征的GAP蛋白中的GAP活性的点突变(R737A)时，全长CARMIL-GAP才能在细胞中过表达。和CARMIL一样，CARMIL-GAP定位于肌动蛋白丰富的结构，在营养细胞和饥饿的发育细胞中都有表达。通过同源重组建立的CARMIL-GAP零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、运动和趋化聚集。重要的是，GFP-FL CARMIL-GAP在CARMIL-GAP缺失细胞中的表达挽救了细胞吞噬和趋化聚集的缺陷。利用GAP结构域进行的下拉光谱分析表明，CARMIL-GAP与Rac 1a结合，Rac 1a是Dictyostelials中表达的11种Rac亚型之一。目前的努力主要集中在展示CARMIL-GAP在体内对RAC 1a的调控，以及确定CARMIL-GAP中哪些结构域(例如GAP结构域、CPI结构域)对其功能最关键。