Regulation of ADP-ribosylation factor

ADP-核糖基化因子的调节

• 批准号: 10262023
• 负责人: Paul A Randazzo
• 金额: $ 184.17万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262023
• 关键词: ADP-Ribosylation Factors; Actins; Affect; Ankyrin Repeat; Area; Behavior; Binding; Biochemical; Biochemistry; Biology; Bundling; Catalysis; Cell Line; Cell Proliferation; Cell fusion; Cell physiology; Childhood Rhabdomyosarcoma; Collaborations; Cytoskeleton; Defect; Dorsal; Endocytosis; Exocytosis; F-Actin; Focal Adhesions; GTP Binding; GTP-Binding Proteins; GTPase-Activating Proteins; Goals; Guanosine Triphosphate; Hydrolysis; Ligands; Link; Lipid Binding; Malignant Neoplasms; Mediating; Membrane; Microfilaments; Mitosis; Modeling; Myoblasts; N-terminal; Neoplasm Metastasis; Oncoproteins; PH Domain; Pathologic Processes; Pathway interactions; Pediatric Oncology; Phosphatidylinositols; Proline; Property; Regulation; Resolution; Rhabdomyosarcoma; SH3 Domains; Signal Pathway; Signal Transduction; Stress Fibers; Structure; Tertiary Protein Structure; Therapeutic; Tumor Cell Invasion; Work; cancer cell; cell behavior; cell motility; childhood sarcoma; enzyme activity; member; outcome forecast; overexpression

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.

ADP核糖化因子(ARF)是RAS超家族中的一员，它协调膜和肌动蛋白重塑，参与细胞运动、内吞和胞吐、有丝分裂等一系列细胞功能，是肿瘤细胞侵袭和转移等病理过程的核心。在我们对Arf调控的研究中，我们发现了Arf GTP酶激活蛋白(GAP)，它促进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，它参与了分化的调节，也被认为是癌细胞行为的调节因子，包括侵袭和转移。此外，ASAP1在一些癌症中过表达，包括儿童横纹肌肉瘤，并且在一些癌症中过表达与不良预后相关，这促使我们最近关注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。在过去的一年里，我们发现ASAP1的BAR和PH结构域直接与F-肌动蛋白结合，从而驱动F-肌动蛋白的捆绑。在正在进行的研究中，我们正在确定ASAP1驱动的肌动蛋白捆绑对应力纤维、内翻足和圆形背褶皱中肌动蛋白重塑的贡献。在与儿科肿瘤科Marielle Yohe博士合作的第三个领域，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是研究ASAP1在癌细胞中功能的理想模型。首先，对于其他癌症，ASAP1在融合阴性的横纹肌肉瘤中过度表达。其次，虽然ASAP1已被发现影响未转化细胞的分化和癌细胞的增殖，但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。