Novel Functions for Ras Family GTPases

描述(由申请人提供)： 项目摘要：RAS蛋白在生物学的许多方面发挥着核心作用。这些单体GTP酶在失活的GDP结合状态和激活的GTP结合状态之间循环。RAS的突变激活发生在大约30%的人类肿瘤中，并捕获处于GTP结合状态的RAS，导致致癌转化。因此，RasGTP被认为是该分子唯一具有生物学意义的形式。然而，越来越多的证据表明，情况可能并非如此。我们发现，无核苷酸RAS(nf-RAS)作为RasGDP向RasGTP转变的中间产物，对磷脂酰肌醇3-激酶IIbeta类(PI3KC2)具有负性调节作用。这些发现对于理解RAS介导的信号传递和转化具有深远的意义。我们认为，nf-RAS与PI3KC2等靶标结合，与这些靶标的相互作用导致RAS和相关靶标的相互抑制。我们的模型进一步预测，RAS的致癌激活导致这些靶标的抑制丧失，从而导致它们的激活与RasGTP结合。因此，我们的发现指出了一类新的分子，对RAS驱动的肿瘤发生很重要，但不结合激活的RAS。这项提议的目的是通过以下三个目的来描述nf-RAS在细胞信号和转化中的这一新角色。在目标1中，我们将使用分子和生化方法相结合的方法来确定nf-RAS与PI3KC2相互作用的分子基础，以绘制RAS与PI3KC2的相互作用图。此外，我们还将确定交叉素支架在调节n-RAS：PI3KC2？复合体中的作用。在目标2中，我们将确定体内处于无核苷酸状态的RAS的比例。最后，在目标3中，我们提出了两种方法来识别nf-RAS的额外靶点，并测试这些靶点在肿瘤发生模型中的重要性。鉴于RAS突变在人类癌症中的普遍存在，了解RAS在肿瘤发生中的作用机制至关重要。我们的工作代表了RAS生物学中一个全新的概念，它定义了一类参与RAS介导的信号和转换的新靶点。这些研究将为RAS介导的肿瘤发生机制提供新的见解，因此具有很高的翻译意义。这项工作将有益于 退伍军人和普通民众都患有癌症。然而，据报道，某些癌症的发病率，如肺癌和胰腺癌，后者的RAS突变发生率很高(90%)，在退伍军人中高出5-7倍，这使得这些研究与退伍军人特别相关。

DESCRIPTION (provided by applicant): Project Summary: Ras proteins play a central role in many aspects of biology. These monomeric GTPases cycle between an inactivate GDP-bound state and an active GTP-bound state. Mutational activation of Ras occurs in approximately 30% of human tumors and traps Ras in the GTP-bound state, leading to oncogenic transformation. Thus, RasGTP has been considered the only biologically important form of the molecule. However, accumulating evidence suggests this may not be the case. We have discovered that nucleotide-free Ras (nf-Ras), formed as an intermediate in the transition from RasGDP to RasGTP, negatively regulates phosphatidyl-inositol 3-kinase, class IIbeta (PI3KC2¿). These finding have profound implications for the understanding of Ras-mediated signaling and transformation. We propose that nf-Ras binds targets such as PI3KC2¿ and that interaction with these targets leads to the mutual inhibition of both Ras and the associated target. Our model further predicts that oncogenic activation of Ras leads to loss of repression of these targets resulting in their activation withou binding RasGTP. Thus, our findings point to a new class of molecules important for Ras-driven tumorigenesis, yet do not bind activated Ras. The goal of this proposal is to characterize this novel role of nf-Ras in cellular signaling and transformation through the following three aims. In Aim 1, we will determine the molecular basis for the interaction of nf-Ras with PI3KC2¿ using a combination of molecular and biochemical approaches to map the interaction of Ras with PI3KC2¿. In addition, we will determine the role of the intersectin scaffold in regulation of the n-Ras: PI3KC2¿ complex. In Aim 2, we will determine the fraction of Ras that resides in the nucleotide-free state in vivo. Finally, in Aim 3 we propose two approaches to identify additional targets of nf-Ras and test the importance of these targets in models of oncogenesis. Given the prevalence of Ras mutations in human cancers, it is critical to understand the mechanisms through which Ras contributes to tumorigenesis. Our work represents an entirely new concept in Ras biology that defines a new class of targets that participate in Ras-mediated signaling and transformation. These studies will provide new insights into the mechanisms of Ras-mediated tumorigenesis and are therefore of high translational significance. This work will be beneficial to Veterans as well as the general population, both of which suffer from cancer. However, the incidence for certain cancers, such as lung and pancreatic cancer the latter of which has a high incidence of Ras mutations (<90%), has been reported to be up to 5-7x higher in Veteran populations making these studies particularly relevant to Veterans.