Regulation of the SNF1/AMPK Pathway in Yeast

描述（由申请人提供）：高度保守的SNF 1/AMP活化蛋白激酶（AMPK）是真核生物中的中心能量调节因子。在人类中，AMPK参与疾病的发病机制，包括2型糖尿病、肥胖和癌症，并且AMPK是抗糖尿病药物的靶点。酵母SNF 1通路为理解人类通路提供了范例。我们建议继续在酵母系统中对SNF 1/AMPK途径进行功能分析，这提供了强大的遗传学优势。SNF 1/AMPK是一种异源三聚体，在代谢和其他应激反应中，通过催化亚基上活化环的磷酸化而活化。越来越多的证据表明蛋白磷酸酶在调节SNF 1/AMPK中的重要性，但涉及的磷酸酶和调节去磷酸化的机制尚不清楚。我们建议研究阐明磷酸酶在SNF 1通路中的作用。我们最近发现，除了Reg 1-Glc 7蛋白磷酸酶1（PP 1），2A型样磷酸酶Sit 4在SNF 1通路中也有作用。我们提出了遗传和生物化学方法来确定这两种磷酸酶是否一起负责SNF 1在高水平葡萄糖生长过程中的去磷酸化，并解决它们在不同应激反应中的作用。这些磷酸酶与SNF 1的相互作用将在不同的条件下进行研究。将评估三个SNF 1亚基在确定每种磷酸酶结合特异性中的作用，并测试阻止激活环去磷酸化的SNF 1的各种改变对磷酸酶相互作用的影响。拟议的研究应提供深入了解在不同条件下控制磷酸酶功能的机制。最后，重要的是要确定所有主要的磷酸酶调节SNF 1途径。有证据表明Ptc 1是一个有前途的候选者，我们计划评估其在SNF 1调控中的作用。我们还将评估其他几种候选酶的作用，并在适当的情况下寻找其他磷酸酶。拟议的研究将为SNF 1/AMPK通路的一般相关性调节机制提供新的见解，并将为识别AMPK的人类磷酸酶的努力提供信息。我们进一步预计，这项工作将指出新的策略，通过靶向磷酸酶及其与AMPK的相互作用来激活激酶，从而预防代谢紊乱和癌症。 公共卫生相关性：高度保守的SNF 1/AMPK通路对于控制代谢和能量稳态是必不可少的，并且在人类中在2型糖尿病、肥胖、癌症和遗传性心脏病中起作用。拟议中的对这一途径调控的研究将为人类疾病提供深入了解，并为治疗药物的开发确定新的靶点。

DESCRIPTION (provided by applicant): The highly conserved SNF1/AMP-activated protein kinase (AMPK) is a central energy regulator in eukaryotes. In humans, AMPK is implicated in the pathogenesis of disease, including type 2 diabetes, obesity and cancer, and AMPK is a target of anti-diabetic drugs. The yeast SNF1 pathway has provided a paradigm for understanding the human pathway. We propose to continue our functional analysis of the SNF1/AMPK pathway in the yeast system, which offers the advantages of powerful genetics. SNF1/AMPK, a heterotrimer, is activated by phosphorylation of the activation loop on the catalytic subunit in response to metabolic and other stress. Accumulating evidence points to the importance of protein phosphatases in regulating SNF1/AMPK, but the phosphatases that are involved and the mechanisms regulating dephosphorylation are not well understood. We propose studies to elucidate the roles of phosphatases in the SNF1 pathway. We recently found that, in addition to Reg1-Glc7 protein phosphatase 1 (PP1), type 2A-like phosphatase Sit4 has a role in the SNF1 pathway. We propose genetic and biochemical approaches to determine whether these two phosphatases together are responsible for dephosphorylation of SNF1 during growth on high levels of glucose and to address their roles in different stress responses. The interactions of these phosphatases with SNF1 will be investigated under different conditions. The roles of the three SNF1 subunits in determining binding specificity for each phosphatase will be assessed, and various alterations of SNF1 that prevent dephosphorylation of the activation loop will be tested for effects on interactions with phosphatases. The proposed studies should provide insight into mechanisms that control phosphatase function under different conditions. Finally, it will be important to identify all the major phosphatases that regulate the SNF1 pathway. Evidence suggests Ptc1 as a promising candidate, and we plan to assess its role in regulation of SNF1. We will also assess the roles of several other candidates, and, if appropriate, undertake a search for other phosphatases. The proposed studies will provide new insights into regulatory mechanisms of general relevance for SNF1/AMPK pathways and will inform efforts to identify human phosphatases for AMPK. We further anticipate that this work will point to new strategies for preventive interventions for metabolic disorders and cancer, by targeting phosphatases and their interactions with AMPK to activate the kinase. PUBLIC HEALTH RELEVANCE: The highly conserved SNF1/AMPK pathway is essential for control of metabolism and energy homeostasis, and in humans has roles in type 2 diabetes, obesity, cancer, and hereditary heart disease. The proposed studies on regulation of this pathway will provide insight into human disease and identify new targets for development of drugs for treatment.