Regulation of ADP-ribosylation factor

ADP-核糖基化因子（Arfs）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，包括癌细胞侵袭和转移所不可或缺的。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灶性粘连。与这些生物化学活性一致，它涉及调节分化和迁移，也涉及癌症侵袭和转移。此外，ASAP1基因在许多癌症中扩增，并且在许多癌症中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ASAP 1。我们研究了ASAP 1生物化学和生物学的三个方面。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等人发现PH结构域通过与磷酸肌醇结合位点相对的位点直接结合底物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。在过去的两年中，我们已经发现ASAP 1的BAR和PH结构域与F-肌动蛋白的直接结合，其驱动F-肌动蛋白的成束，并且对于应力纤维、侵袭足和圆形背褶中肌动蛋白的重塑是必要的。我们已经开始确定ASAP 1和F-肌动蛋白之间的结合位点。在第三个工作领域，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在癌细胞中功能的模型。首先，与其他癌症一样，ASAP 1在融合阴性横纹肌肉瘤中扩增。第二，ASAP 1被发现影响非转化细胞的分化和癌细胞的增殖，并且融合阴性横纹肌肉瘤在成肌细胞的分化方面有缺陷。在正在进行的研究中，我们开发了一种新的原位异种移植模型，我们正在研究ASAP 1在RMS增殖，侵袭和转移中的作用。

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. 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 ongoing studies using a novel orthotopic xenograft model we have developed, we are examining role of ASAP1 for proliferation, invasion and metastasis of RMS.