Mechanisms of Glucose Signal Transduction in Yeast

描述(由申请人提供)：Snf1蛋白激酶在萌芽酵母酿酒酵母的营养胁迫信号中起着核心作用。作为Snf1的哺乳动物同源物，AMP激活的蛋白激酶(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，二甲双胍是治疗2型糖尿病最广泛的处方药。我们建议研究这种医学上重要的酵母酶在酵母中的调节。 在之前的资助期间，我们开发了一种分析Snf1激活环的磷酸化状态的方法。我们发现，Snf1的磷酸化是激活Snf1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，我们在体内证明了有三种上游激酶(Pak1、Tos3和Elm1)能够激活Snf1。Snf1激活酶的鉴定具有重要意义，因为它立即导致了激活AMPK的哺乳动物激酶的鉴定。 在下一个授权期，我们建议继续研究Snf1信号通路的调控。我们将重点关注这一途径的上游事件，并确定调节三种Snf1激活激酶活性的机制。我们将确定是否有不同形式的Snf1激酶复合体专门用于应对不同的压力。我们将评估我们通过质谱学定位的Snf1激酶复合体中的磷酸化位点的调节意义。最后，生化和遗传分析已经确定了一些Snf1信号通路调节器的候选者。将对这些候选蛋白进行研究，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.