Role of the Snf1 protein kinase in regulating eukaryotic gene expression

描述（由申请人提供）：拟议研究的主要目的是全面了解酿酒酵母Snf1蛋白激酶的功能。这两个特定目的本质上是探索性的，旨在使用蛋白质组学和基因组学方法鉴定由Snf1磷酸化的蛋白质和受Snf1调控的基因。Snf 1是信号传导级联的关键组成部分，该级联协调细胞对酵母中葡萄糖和其他营养素可用性变化的反应。Snf1在结构和功能上与人类AMP激活蛋白激酶AMPK高度相关。AMPK作为细胞内代谢状况的传感器，与糖尿病、肥胖、癌症和心脏病有直接联系。使用Snf1作为模型，我们试图确定由这些重要的代谢和营养传感器调控的蛋白质和基因的全谱。具体目标1是鉴定Snf1的直接蛋白底物。该方法是确定蛋白质磷酸化的变化，在全基因组水平上，发生在快速失活的Snf1。这些实验将采用基因修饰的Snf1衍生物，这些衍生物对化学抑制剂特别敏感或通过蛋白水解有条件地破坏。双向差异凝胶电泳结合质谱将用于鉴定细胞蛋白质之间的磷酸化变化。目标1的第二个目标是直接测试RNA聚合酶II的磷酸化是否受Snf1的调节，这是基于我们的遗传观察的预测。具体目标2是鉴定其转录直接受Snf1影响的基因。该方法是对Snf1功能致敏的酵母菌株进行DNA微阵列研究。在一种情况下，菌株将表达可以快速和有条件地失活的Snf1衍生物。在第二种情况下，菌株将在SNF4中表达功能获得性突变，SNF4是Snf1的正调节因子。目标2的第二个目标是建立一个体外转录试验，以测试Snf1是否可以直接调节RNA聚合酶II转录机制。拟议的研究与人类状况直接相关。值得注意的是，AMPK在糖尿病、肥胖、癌症和心脏病中发挥作用，这些疾病是对公共健康最常见和最致命的威胁。Snf1和AMPK之间及其潜在靶点之间的保守性强烈表明，拟议的研究将为AMPK的功能提供新的见解。

DESCRIPTION (provided by applicant): The primary objective of the proposed research is to provide a comprehensive understanding of the functions of the Saccharomyces cerevisiae Snf1 protein kinase. The two Specific Aims are exploratory in nature and are designed to identify, using proteomic and genomic approaches, proteins that are phosphorylated by and genes that are regulated by Snf1. Snf1 is a critical component of a signal transduction cascade that orchestrates cellular responses to changes in the availability of glucose and other nutrients in yeast. Snf1 is highly related, both structurally and functionally, to the human AMP-activated protein kinase, AMPK. AMPK, as a sensor of the metabolic conditions within cells, has direct links to diabetes, obesity, cancer, and heart disease. Using Snf1 as a model, we seek to identify the full spectrum of proteins and genes regulated by these important metabolic and nutritional sensors. Specific Aim 1 is to identify direct protein substrates of Snf1. The approach is to identify changes in protein phosphorylation, at a genome-wide level, that occur upon rapid inactivation of Snf1. These experiments will employ genetically modified Snf1 derivatives that are specifically susceptible to chemical inhibitors or conditionally destroyed by proteolysis. Two-dimensional difference gel electrophoresis combined with mass spectrometry will be used to identify phosphorylation changes among cellular proteins. A second goal of Aim 1 is to test directly whether the phosphorylation of RNA polymerase II is regulated by Snf1, a prediction based on our genetic observations. Specific Aim 2 is to identify genes whose transcription is directly influenced by Snf1. The approach is to perform DNA microarray studies on yeast strains that are sensitized to Snf1 function. In one case, strains will express Snf1 derivatives that can be rapidly and conditionally inactivated. In a second case, strains will express a gain-of-function mutation in SNF4, a positive regulator of Snf1. A second goal of Aim 2 is to establish an in vitro transcription assay to test whether Snf1 can directly regulate the RNA polymerase II transcription machinery. The proposed studies have direct relevance to the human condition. Remarkably, AMPK has roles in diabetes, obesity, cancer and heart disease, some of the most common and deadly threats to public health. The conservation between Snf1 and AMPK and between their potential targets strongly suggests that the proposed studies will provide new insights into the function of AMPK.