Phosphorylation networks regulated by energy stress in yeast

描述（由申请人提供）：真核生物已经进化出在营养物质可用性和能量消耗变化时维持能量稳态的机制。AMP活化蛋白激酶（AMPK）是一种保守的细胞能量状态传感器，在控制细胞代谢的信号网络中起着关键节点的作用。AMPK活性的抑制与胰岛素抵抗、肥胖、癌症和心脏病有关，并且用于治疗II型糖尿病的主要临床药物是AMPK激活剂。AMPK被细胞能量应激激活，其随后的多个靶蛋白的磷酸化用于增加催化剂和减少能量消耗以维持能量平衡。虽然AMPK的几个关键磷酸化靶点是已知的，但可能还有许多其他底物有待发现。这些研究将集中在Snf 1，AMPK的酵母直系同源物，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识别大量的新型Snf 1底物。通过对鸟枪磷酸化蛋白质组学数据进行基于基序的分析，我们已经确定了大约100种Snf 1的潜在底物。我们将开发用于相对定量这些底物的磷酸化状态的测定方法， 细胞提取物，使用靶向质谱法。在Snf 1的化学遗传抑制后丰度减少的位点被认为是依赖于体内激酶。然后，我们将使用一种新的遗传方法来确定哪些位点被Snf 1直接磷酸化。我们通过基于结构的设计产生了Snf 1突变体，该突变体将其磷酸化受体残基偏好从Ser交换到Thr。通过在底物的磷酸化位点引入补偿突变，我们可以通过突变体Snf 1恢复磷酸化。在体内产生功能性重新工程化的激酶-底物对的能力提供了直接磷酸化的有力证据。我们将通过对新型直接底物的详细功能分析来表征Snf 1依赖的磷酸化网络控制甘油脂质代谢和自噬。将检查预测对人类保守的底物在哺乳动物细胞中通过AMPK的调节。这些研究将为Snf 1和人类AMPK控制细胞代谢以响应营养物质可用性变化的机制提供基本见解。此外，用于这些研究的方法应该也适用于其他激酶，为阐明真核生物中磷酸化依赖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.