G protein regulation by monoubiquitination

描述（由申请人提供）：Ras家族GTP酶的持续信号传导可导致癌症以及发育障碍。最近的研究结果表明，H-和K-Ras是monoubiquitinated，这些修饰的蛋白质积累在GTP结合（激活）状态的细胞。我们的初步数据表明，monoubiquitinated Ras是难治性的GTdR活化蛋白，但在其他方面是完全功能性的。基于这些发现，我们提出，monoubiquitination直接激活Ras，在没有持续的激素刺激或致癌突变。在这里，我们提出了一个全面的（在体内和体外）分析Ras monoubiquitination在酵母。 目标1。单泛素化Ras 1的功能分析。我们最近已经表明，Ras 1，而不是Ras 2，是monoubiquitinated在酵母中。这提供了一个独特的机会来比较两种功能相似但调节方式不同的蛋白质。我们的假设是，monoubiquitination导致Ras 1的持续激活。我们将确定Ras 1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Ras信号传导。为了建立机制，我们将确定monoubiquitination如何影响蛋白质及其体外结合伴侣的生化特性。 目标2. Ras 1单泛素化的动态调节。我们的假设是，Ras 1 monoubiquitination是一个动态调节事件。我们的初步证据表明，Ras 1是monoubiquitinated作为刺激依赖性，磷酸化依赖性反馈机制的一部分。使用现有的基因缺失，我们将确定哪些酶是必要的磷酸化，monoubiquitination和去泛素化的Ras 1。使用纯化的蛋白质，我们将确定哪些酶足以在体外进行每种修饰。 目的3单泛素化Ras 1的下游靶标。酵母基因组编码43个泛素结合结构域（UBD）蛋白，在许多情况下作为细胞内的“泛素受体”。我们的假设是，选择UBD蛋白直接和特异性地与monoubiquitinated Ras 1相互作用，从而改变Ras 1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，我们将确定UBD的目标monoubiquitinated Ras 1，但不是未修饰的Ras 1或Ras 2。 我们的方法整合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有明显相关性的过程。如果成功，我们的实验将揭示（第一次）负责启动，添加，删除和识别结合到Ras的泛素的蛋白质。酵母使用类似于在人类中发现的Ras信号传导装置。因此，更全面地了解Ras信号是如何在酵母中调节的，最终可能会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.