Yeast Genetic Selection for Human AMPK Kinases

说明（由申请人提供）：AMP活化蛋白激酶（AMPK）是哺乳动物细胞的“燃料表”，并作为响应代谢应激的葡萄糖和脂质代谢的主要调节剂。AMPK与2型糖尿病、肥胖症和心脏病有关，它是激素瘦素和脂联素以及抗糖尿病药物二甲双胍的靶点。激活AMPK的上游激酶AMPKKs直到最近才被发现。我们对AMPK的酵母（酿酒酵母）直系同源物Snf1蛋白激酶的上游激酶的研究使我们鉴定出LKB1肿瘤抑制激酶是肝脏中主要的AMPKK。该提案探索了酵母遗传系统的创新用途，以识别其他人类AMPKKs。在缺乏Snf1天然上游激酶的酵母突变细胞中，LKB1的表达激活Snf1。将开发新的遗传测定以利用AMPK和Snf1途径之间的这种功能保守性，以便容易地检测和选择酵母细胞中的AMPKK功能。该测定实用性的关键标准是从肝脏cDNA表达文库中回收表达LKB 1的cDNA克隆的能力。来自AMPK发挥重要作用的组织的人cDNA表达文库随后将用于选择在该酵母遗传测定中激活Snf1的激酶。为了验证候选激酶是否为潜在的AMPKK，将验证其磷酸化和激活AMPK的能力。AMPKK可能对特定组织具有特异性，并可能对特定代谢应激条件产生的特定激素或信号作出反应。因此，AMPKK可能是比AMPK更特异的治疗糖尿病和肥胖症的治疗靶点。

DESCRIPTION (provided by applicant): AMP-activated protein kinase (AMPK) is the "fuel gauge" of mammalian cells and serves as a major regulator of glucose and lipid metabolism in response to metabolic stress. AMPK is implicated in type 2 diabetes, obesity, and cardiac disease, and it is a target of the hormones leptin and adiponectin and the antidiabetic drug metformin. The upstream kinases that activate AMPK, the AMPKKs, remained unknown until recently. Our studies of the upstream kinases of the yeast (Saccharomyces cerevisiae) ortholog of AMPK, Snf1 protein kinase, led us to identify the LKB1 tumor suppressor kinase as the major AMPKK in liver. This proposal explores an innovative use of the yeast genetic system to identify other human AMPKKs. In yeast mutant cells lacking the native upstream kinases for Snf1, expression of LKB1 activates Snf1. New genetic assays will be developed to take advantage of this functional conservation between the AMPK and Snf1 pathways in order to readily detect and select for AMPKK function in yeast cells. The key criterion for utility of the assay will be ability to recover cDNA clones expressing LKB1 from a liver cDNA expression library. Human cDNA expression libraries from tissues in which AMPK plays important roles will then be used to select for kinases that activate Snf1 in this yeast genetic assay. To validate candidate kinases as potential AMPKKs, their ability to phosphorylate and activate AMPK will be verified. AMPKKs may be specific to particular tissues and may respond to specific hormones or signals generated by particular metabolic stress conditions. Hence, AMPKKs are potentially more specific therapeutic targets for treatment of diabetes and obesity than AMPK.