Control of actin assembly in cells through regulation of Capping Protein

CARMILs （Capping protein Arp2/3 Myosin I Linker）是一种从原生动物到人类表达的1000个残基多结构域支架蛋白，其结合Capping蛋白（CP）并降低其对肌动蛋白丝刺端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，因为它们与Rac GEF Trio表现出遗传和物理相互作用（Liang et al MBoC 2009; Vanderzalm et al.）。Dev. 2009)，缺乏CARMIL-2的T细胞在CD28 （T细胞信号的主要共受体）下游表现出深刻的信号阻断（Liang et al . Nat. Immunol. 2013）。在之前的工作中（Jung et al JCB 2001），我们发现Dictyostelium CARMIL结合CP、Arp2/3复合物和myosin I（通过其SH3结构域），并且它是行动蛋白依赖过程（如趋化性和微胞吞噬）的必要条件。在这里，我们描述了CARMIL-GAP的初步研究，第二种Dictyostelium CARMIL除了包含所有正常的CARMIL结构域（包括cp结合的CPI结构域）外，还包含一个130残基插入，通过同源性，它是Rho-GTPases的GTPase激活（GAP）结构域。这个结构域可能是功能性的，因为全长CARMIL-GAP只有在其GAP结构域包含一个点突变（R737A）时才能过表达，该突变（R737A）阻断了所有表征的GAP蛋白的GAP活性。此外，GAP结构域下拉的质谱分析表明，CARMIL-GAP结合Rac异构体Rac1A。目前的工作是在体外证明CARMIL-GAP对Rac1A具有GAP活性。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发育细胞中表达。一致地，同源重组产生的CARMIL-GAP零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、运动和趋化聚集。重要的是，这些缺陷是特异性的，因为在CARMIL-GAP缺失的细胞中表达GFP-FL CARMIL-GAP可以挽救它们。最后，对缺乏GAP活性或结合CP能力的CARMIL-GAP版本的CARMIL-GAP无效细胞的修复表明，尽管这两个结构域都对CARMIL-GAP功能有重要贡献，但GAP结构域发挥的作用更大。

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.