Regulation of ADP-ribosylation factor

ADP-核糖基化因子的调节

• 批准号: 10702294
• 负责人: Paul A Randazzo
• 金额: $ 192.01万
• 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/10702294
• 关键词: ADP-Ribosylation Factors; Actins; Affect; Ankyrin Repeat; Area; Behavior; Binding; Binding Sites; Biochemical; Biochemistry; Biology; Bundling; Catalysis; Cell Line; Cell Proliferation; Cell fusion; Collaborations; Cytoskeleton; Defect; Dorsal; F-Actin; Focal Adhesions; GTP Binding; GTP-Binding Proteins; GTPase-Activating Proteins; Genes; Goals; Guanosine Triphosphate; Hydrolysis; In Vitro; Ligands; Link; Lipid Binding; Malignant Neoplasms; Mediating; Membrane; Microfilaments; Modeling; Mus; Myoblasts; N-terminal; Neoplasm Metastasis; Oncoproteins; PH Domain; Pathologic; Pathway interactions; Phosphatidylinositols; Prognosis; Proline; Property; RAS Superfamily Proteins; Regulation; Resolution; Rhabdomyosarcoma; SH3 Domains; Signal Pathway; Signal Transduction; Site; Squamous Cell; Stress Fibers; Structure; Tertiary Protein Structure; Therapeutic; Work; cancer cell; cell behavior; childhood sarcoma; enzyme activity; in vivo; member; migration; tumor progression

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.

ADP-核糖基化因子（Arfs）是Ras超家族的成员。他们协调 膜和肌动蛋白重塑，它们是正常和病理细胞行为的组成部分， 包括癌细胞侵袭和转移。我们发现Arf GTP酶激活 蛋白（GAP），其促进与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，ASAP 1，由BAR，PH，Arf GAP，Ankyrin组成， 重复、富含脯氨酸、E/DLPPKP重复和SH 3结构域。它调节肌动蛋白的重塑 细胞骨架和相关的局灶性粘连。与这些生物化学活动相一致， 与调节分化和迁移有关， 在癌症侵袭和转移中的作用。此外，ASAP 1基因在许多细胞中扩增， 癌症和表达与许多癌症的不良预后相关。的潜在 与癌症进展的联系促使我们关注ASAP 1。我们从三个方面研究了ASAP 1 生物化学和生物学，在过去一年中，这三个领域都取得了进展。首先，我们 致力于确定Arf GAP结构域调节催化的机制。在 去年，我们发现PH结构域是催化口袋的组成部分， Arf GAP结构域的功能所必需的，并与R.安德鲁·伯德， 已经发现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。我们目前正在扩大 以原子分辨率定义机制的工作。在第二个研究领域，我们 研究ASAP 1结合的癌蛋白和它所引起的肌动蛋白重塑之间的联系， 调解。在过去的两年中，我们已经发现了BAR和PH结构域的直接结合， ASAP 1转化为F-肌动蛋白，它驱动F-肌动蛋白的捆绑，并且对于重塑细胞是必要的 肌动蛋白在应力纤维，侵入足和圆形背褶。我们已经开始定义 ASAP 1和F-肌动蛋白之间的结合位点。在第三个工作范畴，我们正研究 ASAP 1对融合阴性横纹肌肉瘤（FN-RMS）行为的贡献， ASAP 1在癌细胞中的功能。首先，与其他癌症一样，ASAP 1在 融合阴性横纹肌肉瘤第二，已经发现ASAP 1影响两种分化 非转化细胞和癌细胞的增殖和融合阴性横纹肌肉瘤 在成肌细胞的分化方面有缺陷。在过去的一年里，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， 对成肌细胞和融合阴性横纹肌肉瘤的作用存在差异。纳入不断 在体外和体内研究中，包括活体研究，我们正在研究这些 差异影响分化、增殖、侵袭和转移。