Mechanisms of Glucose Signal Transduction in Yeast

描述（由申请人提供）：Snf1蛋白激酶在芽殖酵母（酿酒酵母）的营养胁迫信号传导中发挥核心作用。作为Snf1的哺乳动物同源物，AMP活化蛋白激酶（AMPK）在哺乳动物细胞中起着同样重要的作用。最近AMPK被证明是二甲双胍的靶点，二甲双胍是用于治疗2型糖尿病的最广泛的处方药。我们建议研究酵母中这种医学上重要的酶的调节。 在上一个资助期，我们开发了一种测定Snf1激活环磷酸化状态的方法。我们发现，Snf1的磷酸化是激活Snf1激酶的关键步骤，并且Snf1的磷酸化是由一个不同的上游激酶催化的。最近，我们能够证明，有三个上游激酶（Pakl，Tos 3和Elm 1），能够在体内激活Snf 1。Snf1激活激酶的鉴定非常重要，因为它立即导致了激活AMPK的哺乳动物激酶的鉴定。 在下一个资助期间，我们建议继续我们的研究Snf1激酶信号通路的调节。我们将专注于上游事件在这一途径，并确定调节三个Snf1激活激酶的活性的机制。我们将确定是否有不同形式的Snf1激酶复合物，专门用于响应不同的压力。我们将评估我们通过质谱绘制的Snf1激酶复合物中磷酸化位点的调控意义。最后，生化和遗传分析已经确定了一些候选人的调节Snf1激酶信号通路。这些候选蛋白将被研究，以确定它们是否与Snf1激酶相关并调节其活性。

DESCRIPTION (provided by applicant): The Snf1 protein kinase plays a central role in nutrient stress signaling in the budding yeast, Saccharomyces cerevisiae. The mammalian homologue of Snf1, the AMP-activated protein kinase (AMPK) plays an equally important role in mammalian cells. Recently AMPK was shown to be the target of metformin, the most widely prescribed drug used to treat type 2 diabetes. We propose to study the regulation of this medically important enzyme in yeast. In the previous grant period we developed an assay for the phosphorylation state of the Snf1 activation loop. We showed that phosphorylation of Snf1 was a critical step for the activation of the Snf1 kinase and that the phosphorylation of Snf1 was catalyzed by a distinct upstream kinase. Recently, we were able to demonstrate that there are three upstream kinases (Pakl, Tos3 and Elm1) that are capable of activating Snf1 in vivo. The identification of the Snf1 activating kinases was highly significant because it immediately led to the identification of the mammalian kinase that activates AMPK. In the next grant period we propose to continue our studies of the regulation of the Snf1 kinase signaling pathway. We will focus on upstream events in this pathway and determine the mechanism that regulate the activity of the three Snf1-activating kinases. We will determine whether there are distinct forms of the Snf1 kinase complex that are specialized for response to different stresses. We will assess the regulatory significance of phosphorylation sites in the Snf1 kinase complex that we have mapped by mass spectrometry. Finally, biochemical and genetic analyses have identified a number of candidates for regulators of the Snf1 kinase signaling pathway. These candidates proteins will be studied to determine if they associate with and regulate the activity of the Snf1 kinase.