Characterization of growth factor-coupled signaling

描述（由申请人提供）：我们在GM 040654支持下的工作的计划目标是阐明由EGFR和/或ErbB 2-Rho GT3信号传导轴触发的新生物学活性，该信号传导轴在转化表型的形成和维持中至关重要。这些努力涉及生物化学，细胞生物学和结构方法的组合，以及最近的小鼠模型。最近的工作集中在我们在癌细胞中发现了一种令人兴奋的信号传导结果，该结果导致关键代谢酶谷氨酰胺酶C（GAC）的激活，该酶催化谷氨酰胺水解为谷氨酸盐和氨。这代表了提高谷氨酰胺代谢的关键步骤，谷氨酰胺代谢与糖酵解的变化（“瓦尔堡效应”）一起，现在被广泛认为是恶性转化所必需的。因此，了解负责触发这些代谢变化的调节机制对癌症生物学和药学界具有极大的兴趣。鉴于其在谷氨酰胺代谢中的重要作用，GAC可能在许多生物学背景下受到调控。在这次更新申请中，我们将重点关注GAC如何在乳腺癌细胞中被激活，以及其满足“谷氨酰胺成瘾”和恶性状态发展的后果。我们还将描述我们最近发现的一组新的变构GAC抑制剂的作用机制。它们负责突出不同信号蛋白与谷氨酰胺代谢之间的联系，从而可能为阻断癌症进展提供新的治疗策略。这些努力将通过以下研究路线进行：1）定义导致乳腺癌细胞中GAC活化的磷酸化事件。我们将确定参与的蛋白激酶和负责触发乳腺癌细胞系以及从人乳腺肿瘤样品中分离的细胞中GAC激活的基本磷酸化位点。2)确定一类新的阻断GAC活化的变构抑制剂的机制基础。我们将确定一种新发现的称为968的GAC变构抑制剂及其相关化合物如何与该酶结合并阻断其活化。特别是，我们想确定这类抑制剂是否优先结合到GAC的非活性状态和/或阻断GAC磷酸化的能力，从而阻止其激活的关键步骤。3)确定GAC活化在转化和肿瘤形成中的作用。我们将使用细胞和小鼠肿瘤模型来确定这种关键代谢酶的激活是否足以驱动转化，或者其主要作用是否是与致癌信号蛋白一起工作以维持转化状态。这些研究应该提供新的见解，如何重要的代谢变化在癌细胞中表现出来，以及确定新的目标，可能是真实的价值，作为网站的干预对这种疾病。

DESCRIPTION (provided by applicant): The programmatic goals of our work supported by GM040654 have been to elucidate novel biological activities triggered by the EGFR- and/or ErbB2-Rho GTPase signaling axes critical in the development and maintenance of the transformed phenotype. These efforts have involved a combination of biochemical, cell biological, and structural approaches, and more recently mouse models. Recent work has focused on our discovery of an exciting signaling outcome in cancer cells that results in the activation of a key metabolic enzyme, glutaminase C (GAC), which catalyzes the hydrolysis of glutamine to glutamate plus ammonia. This represents a critical step for elevating glutamine metabolism, which together with changes in glycolysis (the "Warburg effect"), is now widely recognized to be essential for malignant transformation. Thus, understanding the regulatory mechanisms responsible for triggering these metabolic changes is of great interest to the cancer biology and pharmaceutical communities. Given its important role in glutamine metabolism, GAC is likely to be regulated in a number of biological contexts. In this renewal application, we will focus on how GAC is activated in breast cancer cells and its consequences for satisfying their "glutamine addiction" and the development of the malignant state. We also will delineate the mechanisms of action of a novel group of allosteric GAC inhibitors that we recently discovered. They were responsible for highlighting the connections between different signaling proteins and glutamine metabolism, and thus potentially offer new therapeutic strategies for blocking cancer progression. These efforts will be pursued through the following lines of investigation: 1) Defining the phosphorylation event(s) that leads to GAC activation in breast cancer cells. We will identify the protein kinase(s) involved and the essential phosphorylation site(s) responsible for triggering GAC activation in breast cancer cell lines as well as in cells isolated from human breast tumor samples. 2) Determining the mechanistic basis for a new class of allosteric inhibitors that block GAC activation. We will establish how a newly identified allosteric inhibitor of GAC called 968, and related compounds, bind to the enzyme and block its activation. In particular, we want to determine whether this class of inhibitors binds preferentialy to the inactive state of GAC and/or blocks the ability of GAC to be phosphorylated and thus prevents a key step in its activation. 3) Determining the role of GAC activation in transformation and tumor formation. We will determine whether the activation of this key metabolic enzyme is sufficient to drive transformation, or if its primary role is to work together with oncogenic signaling proteins to sustain the transformed state, using cell and mouse tumor models. These studies should provide new insights into how important metabolic changes in cancer cells are manifested, as well as identify novel targets that could be of real value by serving as sites of intervention against this disease.