Spatio-temporal regulation of ARF signaling in vesicle formation

项目摘要/摘要 囊泡双向转运在所有真核细胞向分泌细胞器输送蛋白质方面都是至关重要的。 和细胞表面，并从细胞中释放。据估计，30%的哺乳动物蛋白质组 必须通过分泌通道。GBF1(高尔基局部化Brefeldin A敏感因子1)是一个关键 从高尔基到ER的逆行交通的调节器，以及GBF1活动对于 分泌途径的建立和维持。GBF1属于一个大鸟嘌呤家族 核苷酸交换因子(GEF)是一种促进ARF上GDP/GTP交换的酶 小分子RAS样GTP酶亚家族。GBF1介导的ARF激活是形成血管内皮细胞瘤所必需的 逆行COPI囊泡，而GBF1代表COPI囊泡形成的上游调节因子，因为它 通过限制ARF的激活来决定囊泡形成的时间和位置。然而，尽管关键是 尽管GBF1在细胞内稳态中的重要性，但我们仍然不知道GBF1本身在细胞中是如何调节的。 具体地说，我们不知道信元如何向GBF1发出信号，通知它蜂窝需要逆行流量 又有什么机制确保GBF1只在正确的时间启动COPI囊泡的形成 去对地方了。这项提议旨在阐明这一谜团。我们将检验这样的假设：细胞有 抑制GBF1活性以防止虚假ARF激活的机制，但在 响应指示逆行交通的信号通路的时间和位置受限的方式 需求。我们的目标是确定细胞如何转换它们对ARF激活和膜运输的需求 变成了空间和时间受限的GBF1函数。我们提出了三个具体目标来确定流程和 调节GBF1的信号通路在所有细胞中的基本交通路线中发挥作用，逆行COPI 对分泌途径的动态平衡至关重要的交通。在目标1中，我们将确定这些机制 通过识别GbF1中的固有靶向信息，选择性地将GBF1靶向高尔基膜 GBF1和标记GBF1募集的膜部位的膜组件。在目标2中，我们将 通过评估GbF1在膜上的作用，确定调节膜上GBF1催化活性的机制 磷脂酰肌醇磷酸(PIP)在调节GBF1催化活性中的作用。在目标3中，我们将确定 通过定义角色，协调GBF1与COPI流量需求的信号通路发挥作用 GBF1膜结合催化中KDEL-R活化及PKA和SFK信号转导途径的研究 活动。广泛表达，对细胞和生物体健康至关重要 培养细胞的枯竭会导致死亡，而老鼠或果蝇的基因敲除会导致胚胎死亡。 GBF1在病理环境中也很重要，因为它对胶质母细胞瘤细胞的迁移是必不可少的 以及人类致病肠道病毒的复制。我们的研究将提供关键的新知识 GBF1在基本细胞生理学中的调节，并将为治疗设计提供策略信息 在病理背景下控制GBF1介导的事件的干预。

Project Summary/Abstract Bi-directional vesicular transport is vital in all eukaryotic cells to deliver proteins to secretory organelles and the cell surface, and for release from the cell. It is estimated that 30% of the mammalian proteomes must traffic the secretory pathway. GBF1 (Golgi localized Brefeldin A-sensitive Factor1) is a key regulator of retrograde traffic from the Golgi to the ER, and GBF1 activity is required for the establishment and maintenance of the secretory pathway. GBF1 belongs to a family of large Guanine nucleotide Exchange Factors (GEFs) and is an enzyme that facilitates GDP/GTP exchange on the ARF subfamily of small Ras-like GTPases. GBF1-mediated ARF activation is required for the formation of retrograde COPI vesicles, and GBF1 represents an upstream regulator of COPI vesicle formation as it dictates the time and site of vesicle formation by restricting ARF activation. Yet, despite the critical importance of GBF1 in cellular homeostasis, we remain ignorant of how GBF1 itself is regulated in cells. Specifically, we do not know how cells signal to GBF1 to “notify” it of a cellular need for retrograde traffic and what mechanisms ensure that GBF1 initiates COPI vesicle formation only at the right time and the right place. This proposal aims to illuminate this enigma. We will test the hypothesis that cells have mechanisms to inhibit GBF1 activity to prevent spurious ARF activation, but release such inhibition in a time and site-restricted manner in response to a signaling pathway that indicates retrograde traffic demand. Our goal is to define how cells translate their need for ARF activation and membrane transport into space- and time-restricted GBF1 function. We propose 3 specific aims to identify the processes and signaling pathways that regulate GBF1 function in an essential traffic routing in all cells, retrograde COPI traffic that is vital for the homeostasis of the secretory pathway. In Aim 1, we will identify the mechanisms that selectively target GBF1 to Golgi membranes by identifying the intrinsic targeting information within GBF1 and the membrane components that mark membrane sites for GBF1 recruitment. In Aim 2, we will define the mechanisms that regulate GBF1 catalytic activity at the membrane by assessing the role of phosphatidylinositol phosphates (PIPs) in regulating GBF1 catalytic activity. In Aim 3, we will determine the signaling pathways that coordinate GBF1 function with the need for COPI traffic by defining the role of KDEL-R activation and the PKA and SFK pathways on GBF1 membrane association and catalytic activity. GBF1 is ubiquitously expressed and critically important to cell and organismal health; GBF1 depletion from cultured cells causes death and a mouse or Drosophila knockout is embryonic lethal. GBF1 is also important in pathological contexts since it is essential for migration of glioblastoma cells and for replication of human pathogenic enteroviruses. Our studies will provide critical new knowledge of GBF1 regulation in basic cellular physiology and will inform strategies for the design of therapeutic intervention to control GBF1-mediated events in pathological contexts.