Regulation of ADP-ribosylation factor

ADP-核糖基化因子（ADP-ribosylation factors，Arfs）是Ras超家族的成员，参与调节细胞膜和肌动蛋白的重塑，参与细胞运动、胞吞和胞吐、有丝分裂等细胞功能，在肿瘤细胞侵袭和转移等病理过程中起重要作用。在我们对Arfs调控的研究中，我们发现了Arf GTP酶激活蛋白（GAP），其促进与Arf结合的GTP水解，将Arf-GTP转化为Arf-GDP。我们发现的第一个GAP，ASAP 1，由BAR，PH，Arf GAP，Ankyrin重复，脯氨酸丰富，E/DLPPKP重复和SH 3结构域组成。它调节肌动蛋白细胞骨架的重塑和相关的局灶性粘连。与这些生物化学活性一致，它涉及调节分化，并且还涉及作为癌细胞行为（包括侵袭和转移）的调节剂。此外，ASAP 1在许多癌症中过表达，包括儿童横纹肌肉瘤，并且过表达与许多癌症的不良预后相关，这促使我们最近关注ASAP 1。我们研究ASAP 1生物化学和生物学的三个方面，在过去的一年里，这三个方面都取得了进展。首先，我们正在努力确定Arf GAP结构域调节催化的机制。在过去的一年中，我们发现PH结构域是催化口袋的一个组成部分，是Arf GAP结构域功能所必需的。Andrew Byrd等发现PH结构域直接结合底物Arf-GTP的N-末端延伸。我们目前正在扩展工作，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ASAP 1结合的癌蛋白与其介导的肌动蛋白重塑之间的联系。在过去的一年中，我们发现了ASAP 1的BAR和PH结构域与F-肌动蛋白的直接结合，这驱动了F-肌动蛋白的捆绑。在正在进行的研究中，我们正在确定ASAP 1驱动的肌动蛋白捆绑的贡献，在应力纤维，侵袭足和圆形背褶肌动蛋白的重塑。在与儿科肿瘤分支的Marielle Yohe博士合作的第三个工作领域，我们正在研究ASAP 1对融合阴性横纹肌肉瘤（FN-RMS）行为的贡献，FN-RMS是ASAP 1在癌细胞中功能的理想模型。首先，与其他癌症一样，ASAP 1在融合阴性横纹肌肉瘤中过表达。第二，虽然ASAP 1已被发现影响非转化细胞的分化和癌细胞的增殖，融合阴性横纹肌肉瘤在成肌细胞的分化中有缺陷。在过去的一年中，我们发现ASAP 1调节成肌细胞和横纹肌肉瘤细胞系的分化途径，并且对成肌细胞和融合阴性横纹肌肉瘤的影响存在差异。在正在进行的研究中，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 that coordinated membrane and actin remodeling, which integral to a number of cellular functions, including cell movement, endocytosis and exocytosis, and mitosis and are central to pathological processes such as tumor cell invasion and metastasis. In our studies of the regulation of Arfs, 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 has also been implicated as a regulator of cancer cell behaviors, including invasion and metastasis. Furthermore, ASAP1 is overexpressed in a number of cancers, including childhood rhabdomyosarcomas and overexpression correlates with poor prognosis in a number of cancers, which has motivated our recent 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 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 year, we have discovered direct binding of the BAR and PH domain of ASAP1 to F-actin, which drives bundling of the F-actin. In ongoing studies, we are determining the contribution to ASAP1-driven actin bundling to remodeling of actin in stress fibers, invadopodia and circular dorsal ruffles. In a third area of work in collaboration with Dr. Marielle Yohe of Pediatric Oncology Branch, we are examining the contribution of ASAP1 to the behavior of fusion-negative rhabdomyosarcoma (FN-RMS), an ideal model for the function of ASAP1 in cancer cells. First, as for other cancer, ASAP1 is overexpressed in fusion-negative rhabdomyosarcoma. Second, while ASAP1 has been found to affect both differentiation of nontransformed cells and proliferation of cancer cells, 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 studies, we are examining how these differences affect differentiation, proliferation, invasion and metastasis.