Mechanisms of glucose signal transduction in yeast

描述（由申请人提供）：在过去的五十年中，肥胖症的发病率急剧增加。超过一半的美国人超重，超过三分之一的人被归类为临床肥胖。肥胖症的流行对健康的影响表现在2型糖尿病发病率的随之增加。据估计，2500万美国人患有糖尿病，其中95%为2型糖尿病。2型糖尿病最常用的治疗药物靶向AMP活化蛋白激酶（AMPK）。因此，了解AMPK的正常调节机制对人类健康具有重要意义。本研究以面包酵母为模型系统，研究酵母AMPK的调控。在酵母和人类细胞中，AMPK的整体结构及其调节模式是高度保守的。酵母中遗传和生化研究的速度和协同作用使其成为剖析AMPK调节的理想系统。我们实验室过去的研究表明，Snf1（AMPK的酵母名称）的激活至少涉及两个步骤：Snf1激活环的磷酸化和由Snf1复合物的β和γ调节亚基介导的第二个激活步骤。我们已经证明，第一步，控制激活环磷酸化，不受磷酸盐添加水平的调节。令人惊讶的是，正是去磷酸化步骤对细胞能量状态做出了反应。在最近的研究中，我们已经能够概括的调节去磷酸化的Snf1激酶在体外纯化系统。在能量应激条件下，Snf1主要处于活性磷酸化状态。我们可以表明，结合到低能量腺苷酸配体促进形成磷酸酶抗性构象，从而稳定Snf1的活性形式。本提案的具体目标1将使用纯化的Snf1酶表征腺苷酸配体促进磷酸酶抗性状态形成的分子机制。在其他研究中，我们已经表明，配体介导的保护去磷酸化只是一个水平的调节。还必须有其他的监管手段。在具体目标2中，我们将使用遗传和生化筛选来鉴定调节体内Snf1激酶活性的其他组分和信号通路。在最后的目标，我们研究的机制，其中失活Snf1的磷酸酶是针对特定的底物。总之，这些研究将进一步加深我们对AMPK调控的理解，并将有助于开发新的更有效的干预措施来治疗糖尿病。

DESCRIPTION (provided by applicant): The incidence of obesity has increased dramatically over the last fifty years. Over half of all Americans are over-weight and over one third are classified as clinically obese. The health ramifications of this epidemic of obesity are seen in th concomitant increase in the incidence of type 2 diabetes. An estimated 25 million Americans have diabetes with 95% of those being type 2. The most commonly prescribed therapeutic drugs used for type 2 diabetes target the AMP-activated protein kinase (AMPK). Therefore, understanding how AMPK is normally regulated is of great significance to human health. This proposal uses baker's yeast as its model system to study the regulation of the yeast AMPK. In both yeast and human cells, the overall structure of AMPK as well as its modes of regulation is highly conserved. The speed and synergy of genetic and biochemical studies in yeast make this an ideal system to dissect the regulation of AMPK. Past studies from our lab have shown that the activation of Snf1, the yeast name for AMPK, involves at least two steps: phosphorylation of the Snf1 activation loop and a second activation step mediated by the beta and gamma regulatory subunits of the Snf1 complex. We have shown that the first step, control of activation loop phosphorylation, is not regulated at the level of phosphate addition. Surprisingly, it is the dephosphorylation step that responds to cellular energy status. In recent studies we have been able to recapitulate the regulated dephosphorylation of Snf1 kinase in vitro in a purified system. Under conditions of energy stress, Snf1 is primarily in the active phosphorylated state. We can show that binding to low energy adenylate ligands promotes the formation of a phosphatase resistant conformation, thus stabilizing the active form of Snf1. Specific aim 1 of this proposal will characterize the molecular mechanism by which adenylate ligands promote the formation of the phosphatase resistant state using purified Snf1 enzymes. In other studies, we have shown that the ligand-mediated protection from dephosphorylation is only one level of regulation. Additional means of regulation must also be operating. In Specific aim 2, we will use genetic and biochemical screens to identify additional components and signaling pathways that regulate Snf1 kinase activity in vivo. In the final aim we examine the mechanism by which the phosphatase which inactivates Snf1 is targeted to specific substrates. In all, these studies will further our understanding of the regulation of AMPK and will help to develop new and more effective interventions to treat diabetes.