Novel Functions for Ras Family GTPases

描述（由申请人提供）： Ras蛋白在生物学的许多方面发挥着核心作用。这些单体GTPases在一个ADP结合状态和一个活性GTPases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，并将Ras捕获在GTP结合状态，导致致癌转化。因此，RasGTP被认为是该分子的唯一生物学重要形式。然而，越来越多的证据表明，情况可能并非如此。我们已经发现，无核苷酸Ras（nf-Ras），作为从RasGDP到RasGTP过渡的中间体形成，负调节磷脂酰肌醇3-激酶II β（PI 3 KC 2？）。这些发现对于理解Ras介导的信号传导和转化具有深远的意义。我们认为nf-Ras结合靶点如PI 3 KC 2？，与这些靶点的相互作用导致Ras和相关靶点的相互抑制。我们的模型进一步预测Ras的致癌激活导致这些靶点的抑制丧失，导致它们在不结合RasGTP的情况下激活。因此，我们的研究结果指出了一类对Ras驱动的肿瘤发生很重要的新分子，但不结合活化的Ras。该提案的目标是通过以下三个目标来表征nf-Ras在细胞信号传导和转化中的这种新作用。在目的1中，我们将确定nf-Ras与PI 3 KC 2相互作用的分子基础，使用分子和生物化学方法的组合来绘制Ras与PI 3 KC 2的相互作用。此外，我们将确定intersectin支架在调节n-Ras：PI 3 KC 2？复合物中的作用。在目标2中，我们将确定Ras在体内处于无核苷酸状态的分数。最后，在目标3中，我们提出了两种方法来确定nf-Ras的其他靶点，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性，了解Ras促进肿瘤发生的机制至关重要。我们的工作代表了Ras生物学中的一个全新概念，定义了一类参与Ras介导的信号传导和转化的新靶标。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。这项工作将有利于 退伍军人和普通民众都患有癌症。然而，某些癌症的发病率，如肺癌和胰腺癌，其中后者具有Ras突变的高发病率（<90%），据报道退伍军人人群的发病率高出5- 7倍，使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.