Phosphorylation networks regulated by energy stress in yeast

描述(申请人提供)：真核生物已经进化出在营养供应和能量消耗发生变化的情况下维持能量平衡的机制。AMPK是一种保守的细胞能量状态感受器，在控制细胞代谢的信号网络中起着关键作用。抑制AMPK活性与胰岛素抵抗、肥胖、癌症和心脏病有关，治疗II型糖尿病的主要临床药物是AMPK激动剂。AMPK是由细胞能量应激激活的，其随后的多个靶蛋白的磷酸化有助于增加分解代谢，降低能量消耗，以维持能量平衡。虽然AMPK的几个关键的磷酸化靶点是已知的，但很可能还有许多其他底物有待发现。这些研究将集中在酵母AMPK的同源基因Snf1，它长期以来一直是研究AMPK调控和功能的重要模型。我们建议使用新兴的靶向磷酸蛋白质组学方法来鉴定大量新的Snf1底物。通过对鸟枪磷蛋白质组数据的基于基序的分析，我们已经确定了大约100个可能的Snf1底物。我们将开发相对量化这些底物的磷酸化状态的分析方法 使用靶向质谱学方法提取细胞。在化学遗传抑制Snf1之后，丰度降低的位点被认为依赖于体内的激酶。然后，我们将使用一种新的遗传方法来确定哪些位点直接被Snf1磷酸化。我们已经通过基于结构的设计产生了一个Snf1突变体，它将其磷受体残基的偏好从Ser转换为Thr。通过在底物的磷酸化位点引入补偿突变，我们可以恢复突变的Snf1的磷酸化。在体内产生功能性重组的激酶-底物对的能力为直接磷酸化提供了强有力的证据。我们将通过对新的直接底物的详细功能分析来表征控制甘油脂代谢和自噬的依赖于Snf1的磷酸化网络。预计对人类保守的底物将在哺乳动物细胞中受到AMPK的调节。这些研究将为Snf1和人类AMPK控制细胞代谢以响应营养供应变化的机制提供基本的见解。此外，这些研究所使用的方法也应该适用于其他激酶，为阐明真核生物中依赖磷酸化的信号网络提供通用工具。

DESCRIPTION (provided by applicant): Eukaryotes have evolved mechanisms for maintaining energy homeostasis in the face of changes in nutrient availability and energy expenditure. AMP-activated protein kinase (AMPK) is a conserved sensor of cellular energy status, acting as a critical node in signaling networks controlling cellular metabolism. Suppression of AMPK activity has been implicated in insulin resistance, obesity, cancer, and heart disease, and a major clinical drug for treating type II diabetes is an AMPK activator. AMPK is activated by cellular energy stress, and its subsequent phosphorylation of multiple target proteins serves to increase catabolism and decrease energy consumption to maintain energy balance. While several key phosphorylation targets of AMPK are known, it is likely that there are many additional substrates that remain to be discovered. These studies will focus on Snf1, the yeast ortholog of AMPK, which has long served as an important model for studying AMPK regulation and function. We propose to identify a large number of novel Snf1 substrates using emerging targeted phosphoproteomics methodology. Through motif-based analysis of shotgun phosphoproteomics data, we have identified approximately 100 potential substrates of Snf1. We will develop assays for relative quantification of the phosphorylation state of these substrates in cell extracts using a targeted mass spectrometry approach. Sites that decrease in abundance following chemical-genetic inhibition of Snf1 are considered to be dependent on the kinase in vivo. We will then use a novel genetic method to establish which sites are directly phosphorylated by Snf1. We have generated a Snf1 mutant by structure-based design that exchanges its phosphoacceptor residue preference from Ser to Thr. By introducing compensating mutations at the phosphorylation site of substrates, we can restore phosphorylation by mutant Snf1. The ability to generate functional re- engineered kinase-substrate pairs in vivo provides strong evidence of direct phosphorylation. We will characterize Snf1-dependent phosphorylation networks controlling glycerolipid metabolism and autophagy through detailed functional analysis of novel direct substrates. Substrates predicted to be conserved to humans will be examined for regulation by AMPK in mammalian cells. These studies will provide fundamental insight into mechanisms by which Snf1 and human AMPK control cellular metabolism in response to changes in nutrient availability. In addition, the methodology used for these studies should be applicable other kinases as well, providing general tools for elucidating phosphorylation-dependent signaling networks in eukaryotes.