Control of actin assembly in cells through regulation of Capping Protein

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的初步研究，第二个网骨藻CARMIL含有，除了所有正常的CARMIL结构域（包括CP结合CPI结构域），一个130个残基的插入，通过同源性，是一个GTP酶激活（GAP）结构域的Rho-GTP酶。 该结构域可能是功能性的，因为全长CARMIL-GAP只有在其GAP结构域含有阻断所有特征性GAP蛋白中的GAP活性的点突变（R737 A）时才能过表达。 此外，GAP结构域下拉的质谱分析表明CARMIL-GAP结合Rac同种型Rac 1A。 目前的努力旨在体外证明CARMIL-GAP对Rac 1A表现出GAP活性。 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无效细胞系在营养和饥饿细胞中发生的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，这些缺陷是特异性的，因为GFP-FL CARMIL-GAP在CARMIL-GAP缺失细胞中的表达挽救了它们。 最后，用缺乏GAP活性或结合CP能力的CARMIL-GAP版本拯救CARMIL-GAP无效细胞表明，虽然两个结构域都对CARMIL-GAP功能有显著贡献，差距域发挥更大的作用。

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 initial 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 is probably functional given that full length CARMIL-GAP can only be over-expressed if its GAP domain contains a point mutation (R737A) that blocks GAP activity in all characterized GAP proteins. Moreover, mass spec analyses of GAP domain pull-downs indicate that CARMIL-GAP binds the Rac isoform Rac1A. Current efforts are directed at demonstrating in vitro that CARMIL-GAP exhibits GAP activity towards Rac1A. 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 occurring in vegetative and starved cells, including phagocytosis, motility and chemotactic aggregation. Importantly, these defects are specific, as expression of GFP-FL CARMIL-GAP in CARMIL-GAP null cells rescues them. Finally, rescue of CARMIL-GAP null cells with versions of CARMIL-GAP that lack either GAP activity or the ability to bind CP show that while both domains contribute significantly to CARMIL-GAP function, the GAP domain plays a bigger role.