Phosphorylation networks regulated by energy stress in yeast

描述（由申请人提供）：真核生物在面对养分可用性和能量消耗的变化时已经进化出维持能量稳态的机制。amp活化蛋白激酶（AMPK）是细胞能量状态的保守传感器，是控制细胞代谢的信号网络中的关键节点。AMPK活性的抑制与胰岛素抵抗、肥胖、癌症和心脏病有关，治疗II型糖尿病的一种主要临床药物是AMPK激活剂。AMPK被细胞能量应激激活，其随后磷酸化多个靶蛋白，增加分解代谢，减少能量消耗，维持能量平衡。虽然已知AMPK的几个关键磷酸化靶点，但可能还有许多其他底物有待发现。这些研究将重点关注AMPK的酵母同源基因Snf1，它一直是研究AMPK调控和功能的重要模型。我们建议使用新兴的靶向磷酸化蛋白质组学方法鉴定大量新的Snf1底物。通过对散弹枪磷酸化蛋白质组学数据的基序分析，我们已经确定了大约100个Snf1的潜在底物。我们将开发相对定量分析这些底物的磷酸化状态

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.