G protein regulation by monoubiquitination

描述(由申请人提供)：RAS家族GTP酶持续的信号传递可导致癌症和发育障碍。最近的发现表明，H-和K-RAS是单价的，这些修饰的蛋白质在细胞中以GTP结合(激活)的状态积累。我们的初步数据显示，单素化的RAS对GTP酶激活蛋白不起作用，但在其他方面是完全功能的。基于这些发现，我们认为，在没有持续的激素刺激或致癌突变的情况下，单泛素化直接激活RAS。在这里，我们提出了一个全面的(体内和体外)酵母中RAS单泛素化的分析。目的1.单苷化Ras1的功能分析。我们最近已经证明，Ras1，而不是Ras2，在酵母中是一元素化的。这为比较两种功能相似、受不同调控的蛋白质提供了一个独特的机会。我们的假设是，单素化导致Ras1的持续激活。我们将确定Ras1选择性泛素化的结构决定因素。利用泛素化缺陷突变体，我们将确定这种修饰如何影响体内的RAS信号。为了建立机制，我们将在体外确定单一泛素化如何影响蛋白质及其结合伙伴的生化性质。目的2.Ras1单泛素化的动态调节。我们的假设是，Ras1单泛素化是一个动态调节的事件。我们的初步证据表明，Ras1是刺激依赖、磷酸化依赖的反馈机制的一部分。利用现有的基因缺失，我们将确定哪些酶是Ras1的磷酸化、单泛素化和去泛素化所必需的。使用纯化的蛋白质，我们将在体外确定哪些酶对每一种修饰是足够的。目的3个下游靶点单糖化-RAS1。酵母基因组编码43种泛素结合域(UBD)蛋白，在许多情况下，这些蛋白充当细胞内的“泛素受体”。我们的假设是，特定的UBD蛋白直接和单偶联素Ras1特异性地相互作用，从而改变Ras1的运输和信号功能。使用纯化的蛋白质和可用的基因缺失突变体，我们将识别针对单素化Ras1的UBD，而不是针对未修饰的Ras1或Ras2的UBD。我们的方法集成了强大的基因组学和蛋白质组学工具，其中许多工具仅在酵母中可用，以研究与人类健康和疾病明显相关的过程。如果成功，我们的实验将首次揭示负责RAS结合的泛素的启动、添加、移除和识别的蛋白质。酵母菌使用一种与人类相似的RAS信号装置。因此，更全面地了解酵母中RAS信号是如何调节的，最终可能导致从根本上治疗人类RAS相关疾病的新方法。

DESCRIPTION (provided by applicant): Sustained signaling by Ras-family GTPases can lead to cancer as well as developmental disorders. Recent findings reveal that H- and K-Ras are monoubiquitinated and that these modified proteins accumulate in the GTP-bound (activated) state in cells. Our preliminary data reveal that monoubiquitinated Ras is refractory to GTPase activating proteins, but is otherwise fully functional. Based on these findings, we propose that monoubiquitination activates Ras directly, in the absence of a sustained hormone stimulus or oncogenic mutation. Here we propose a comprehensive (in vivo and in vitro) analysis of Ras monoubiquitination in yeast. Aim 1. Functional analysis of monoubiquitinated Ras1. We have shown recently that Ras1, but not Ras2, is monoubiquitinated in yeast. This provides a unique opportunity to compare two functionally similar proteins that are regulated differently. Our hypothesis is that monoubiquitination leads to sustained activation of Ras1. We will identify the structural determinants for selective ubiquitination of Ras1. Using ubiquitination-deficient mutants we will determine how this modification affects Ras signaling in vivo. To establish mechanism, we will determine how monoubiquitination affects the biochemical properties of the protein and its binding partners in vitro. Aim 2. Dynamic regulation of Ras1 monoubiquitination. Our hypothesis is that Ras1 monoubiquitination is a dynamically regulated event. Our preliminary evidence indicates that Ras1 is monoubiquitinated as part of a stimulus-dependent, phosphorylation-dependent feedback mechanism. Using available gene deletions we will determine which enzymes are necessary for the phosphorylation, monoubiquitination and deubiquitination of Ras1. Using purified proteins we will establish which enzymes are sufficient for each modification in vitro. Aim 3 Downstream targets of monoubiquitinated-Ras1. The yeast genome encodes 43 ubiquitin-binding domain (UBD) proteins, which in many cases serve as intracellular "ubiquitin receptors". Our hypothesis is that select UBD proteins interact directly and specifically with monoubiquitinated Ras1, and thereby alter Ras1 trafficking and signaling functions. Using purified proteins and available gene deletion mutants, we will identify the UBDs that target monoubiquitinated Ras1, but not unmodified Ras1 or Ras2. Our approach integrates powerful genomics and proteomics tools, many of which are available only in yeast, to study a process that has clear relevance to human health and disease. If successful, our experiments will reveal (for the first time) proteins responsible for the initiation, addition, removal, and recognition of ubiquitin bound to Ras. Yeast employ a Ras signaling apparatus analogous to that found in humans. Thus a fuller understanding of how Ras signaling is modulated in yeast could eventually lead to fundamentally new approaches to treat Ras-related disease in humans.