Regulation of ADP-ribosylation factor

ADP核糖化因子(ARF)是RAS超家族的成员。它们协调膜和肌动蛋白重塑，这是正常和病理细胞行为所不可或缺的，包括癌细胞的侵袭和转移。我们发现了Arf GTPase激活蛋白(GAP)，它促进与Arf结合的GTP的水解，将Arf-GTP转化为Arf-GDP。我们发现的第一个GAP，ASAP1，由BAR、PH、Arf、Ankyrin重复序列、富含Pro、E/DLPPKP重复序列和SH3结构域组成。它调节肌动蛋白细胞骨架的重塑和相关的局部粘连。与这些生化活性一致的是，它与调节分化和迁移有关，也与癌症的侵袭和转移有关。此外，ASAP1基因在一些癌症中被扩增，并且在一些癌症中的表达与不良预后相关。与癌症进展的潜在联系促使我们关注ASAP1。我们研究了ASAP1生物化学和生物学的三个方面，在过去一年中这三个领域都取得了进展。首先，我们正在努力确定Arf GAP结构域调控催化的机制。在过去的一年里，我们发现PH结构域是催化口袋的组成部分，是Arf间隙结构域发挥功能所必需的，并与R.Andrew Byrd博士合作，发现PH结构域通过与磷脂酰肌醇结合位点相对的位置，直接与底物Arf-GTP的N端延伸结合。我们目前正在扩展这项工作，以定义原子分辨率的机制。在第二个研究领域，我们正在研究ASAP1结合的癌蛋白和它所介导的肌动蛋白重塑之间的联系。在过去的两年里，我们发现了ASAP1的bar和PH结构域与F-肌动蛋白的直接结合，这种结合驱动了F-肌动蛋白的捆绑，并且是应力纤维、内翻足和圆形背褶皱中肌动蛋白重塑所必需的。我们已经开始确定ASAP1与F-肌动蛋白的结合部位。在第三个工作领域，我们正在研究ASAP1在融合阴性横纹肌肉瘤(FN-RMS)行为中的作用，FN-RMS是ASAP1在癌细胞中功能的模型。首先，对于其他癌症，ASAP1在融合阴性的横纹肌肉瘤中扩增。其次，已发现ASAP1既影响未转化细胞的分化，也影响癌细胞的增殖，而融合阴性横纹肌肉瘤在成肌细胞分化方面存在缺陷。在过去的一年里，我们发现ASAP1对成肌细胞和横纹肌肉瘤细胞系的分化途径都有调节作用，并且对成肌细胞和融合阴性横纹肌肉瘤的作用存在差异。在正在进行的体外和体内研究中，包括体内研究，我们正在研究这些差异如何影响分化、增殖、侵袭和转移。

ADP-ribosylation factors (Arfs) are members of the Ras superfamily. They coordinate membrane and actin remodeling, which are integral to normal and pathologic cellular behaviors, including cancer cell invasion and metastasis. We discovered the Arf GTPase-activating proteins (GAPs), which facilitate the hydrolysis of GTP bound to Arf, converting Arf-GTP to Arf-GDP. The first GAP we discovered, ASAP1, is composed of a BAR, PH, Arf GAP, Ankyrin repeat, proline rich, E/DLPPKP repeat and SH3 domains. It regulates remodeling of the actin cytoskeleton and associated focal adhesions. Consistent with these biochemical activities, it has been implicated in regulating differentiation and migration and has also been implicated in cancer invasion and metastasis. Furthermore, the ASAP1 gene is amplified in a number of cancers and expression correlates with poor prognosis in a number of cancers. The potential link to cancer progression has motivated our focus on ASAP1. We study three aspects of ASAP1 biochemistry and biology, with progress in all three areas in the past year. First, we are working towards determining the mechanism of regulated catalysis by the Arf GAP domain. In the past year, we have discovered that the PH domain is an integral part of the catalytic pocket, necessary for function of the Arf GAP domain and, in collaboration with Dr. R. Andrew Byrd, have discovered that the PH domain, through a site opposite the phosphoinositide binding site, binds directly to an N-terminal extension of the substrate Arf-GTP. We are currently extending the work to define mechanism at atomic resolution. In the second area of study, we are examining the link between oncoproteins to which ASAP1 binds and the actin remodeling that it mediates. In the past two years, we have discovered direct binding of the BAR and PH domain of ASAP1 to F-actin, which drives bundling of the F-actin and is necessary for remodeling of actin in stress fibers, invadopodia and circular dorsal ruffles. We have begun to define the binding site between ASAP1 and F-actin. In a third area of work, we are examining the contribution of ASAP1 to the behavior of fusion-negative rhabdomyosarcoma (FN-RMS), a model for the function of ASAP1 in cancer cells. First, as for other cancer, ASAP1 is amplified in fusion-negative rhabdomyosarcoma. Second, ASAP1 has been found to affect both differentiation of nontransformed cells and proliferation of cancer cells and fusion-negative rhabdomyosarcoma has a defect in differentiation of myoblasts. In the past year, we have discovered that ASAP1 regulates differentiation pathways in both myoblasts and rhabdomyosarcoma cell lines and that there are differences in effects on myoblasts and fusion-negative rhabdomyosarcoma. In ongoing in vitro and in vivo studies, including intravital studies, we are examining how these differences affect differentiation, proliferation, invasion and metastasis.