Role of AMP kinase and DAF-16 in Mediating the Effect o*

AMP 激酶和 DAF-16 在介导 o* 效应中的作用

• 批准号: 6649848
• 负责人: MARIA Carmalita ALEXANDER-BRIDGES
• 金额: $ 38.91万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6649848
• 关键词: CHO cells; Caenorhabditis elegans; DNA binding protein; SDS polyacrylamide gel electrophoresis; adenosine monophosphate; autoradiography; caloric dietary content; enzyme activity; gel mobility shift assay; genetic transcription; glucose; hyperinsulinism; insulin; laboratory rat; longevity; mass spectrometry; nutrition related tag; phosphorylation; phosphotransferases; polymerase chain reaction; protein kinase; protein sequence; thin layer chromatography; transcription factor; transfection

DESCRIPTION (provided by applicant): Genetic evidence in worms (C. Elegans) has shown that insulin-like molecules act via PI3 kinase and AKT/protein kinase B to inhibit the function of the forkhead (FKH) transcription factor DAF-16. Insulin signaling mutants with diminished function undergo dauer arrest and show increased longevity and resistance to oxidative stress due to the unimpeded action of DAF-16. Accordingly, DAF-16 has been shown to activate the superoxide dismutase gene (SOD). In mammals, caloric restriction and low insulin signaling has been shown to slow the rate of aging by mechanisms that include increased DNA repair capacity and reduction of oxidative stress. In the presence of low glucose and circulating insulin levels, DAF-16 homologues appear to be transcriptionally active. Several laboratories have shown that in the absence of insulin, mammalian homologues of DAF-16, FKHR, FKHRL1 and AFX activate the transcription of genes that control apoptosis, and gluconeogenesis, and insulin can inhibit this effect. Our goal is to elucidate the mechanisms by which DAF-16 like factors activate transcription in the absence of insulin. Caloric restriction and low glucose activates AMP kinase. We find that AMP kinase can prevent the effect of insulin on DAF-16 in HepG2 cells. Furthermore, AMP kinase can directly phosphorylate DAF-16. We propose to determine whether regulation of DAF-16 by AMP kinase in worms and regulation of its homologue FKHR in mammalian cells, can explain the ability of caloric restriction to slow the aging process. In HepG2 cells, insulin signaling via the AKT sites in DAF-16 inhibits DAF-16 activity. We find that the AKT sites in DAF-16 carry overlapping AMP kinase sites. In Specific Aim 1 of this proposal, we will determine whether AMP kinase regulates DAF-16 activity directly by altering its phosphorylation or indirectly by regulating other elements of the PI3 kinase-signaling pathway. We will examine the effect of AMP kinase on 1) phosph DAF-16 phosphorylation in vitro and in vivo, 2) 14-3-3 binding to DAF-16 in the presence and absence of insulin and 3) the interaction of DAF-16 with other proteins that increase its binding/transcription activity. In Specific Aim II we will determine whether AMP kinase can counteract the effect of insulin signaling to DAF-16 in C. elegans, and prolong life span in the worm. In Specific Aim III, we will examine the effect of caloric restriction and carbohydrate-induced hyperinsulinemia on the activity of AMP kinase and DAF-16 homologues

描述（由申请人提供）：线虫的遗传证据（线虫） 已表明胰岛素样分子通过 PI3 激酶和 AKT/蛋白发挥作用 激酶 B 抑制叉头 (FKH) 转录因子的功能 DAF-16。 功能减弱的胰岛素信号突变体经历了 dauer 逮捕并显示出更长的寿命和对氧化应激的抵抗力 DAF-16的行动不受阻碍。 因此，DAF-16 已被证明 激活超氧化物歧化酶基因（SOD）。 在哺乳动物中，热量限制 低胰岛素信号已被证明可以通过以下方式减缓衰老速度 机制包括增加 DNA 修复能力和减少 氧化应激。 在低血糖和循环胰岛素存在的情况下 水平上，DAF-16 同源物似乎具有转录活性。 一些 实验室表明，在缺乏胰岛素的情况下，哺乳动物的同系物 DAF-16、FKHR、FKHRL1 和 AFX 激活基因转录， 控制细胞凋亡和糖异生，而胰岛素可以抑制这种作用。 我们的目标是阐明 DAF-16 样因子激活的机制 在没有胰岛素的情况下转录。 热量限制和低血糖会激活 AMP 激酶。 我们发现 AMP 激酶可以阻止胰岛素对 HepG2 细胞中 DAF-16 的作用。 此外，AMP激酶可以直接磷酸化DAF-16。 我们建议 确定蠕虫中 AMP 激酶是否对 DAF-16 进行调节以及调节 哺乳动物细胞中其同源物 FKHR 的研究可以解释热量的能力 限制以减缓衰老过程。 在 HepG2 细胞中，胰岛素信号通过 DAF-16 中的 AKT 位点抑制 DAF-16 活性。 我们发现 AKT 站点 DAF-16 中携带重叠的 AMP 激酶位点。 在本次具体目标 1 中 建议，我们将确定 AMP 激酶是否调节 DAF-16 活性 直接通过改变其磷酸化或间接通过调节其他 PI3 激酶信号通路的元件。 我们将检查效果 AMP 激酶对 1) 磷酸 DAF-16 体外和体内磷酸化的影响，2) 14-3-3 在存在或不存在胰岛素的情况下与 DAF-16 结合，并且 3) DAF-16 与其他蛋白质的相互作用，增加其 结合/转录活性。 在具体目标 II 中，我们将确定是否 AMP 激酶可以抵消线虫中胰岛素信号转导至 DAF-16 的影响。 线虫，并延长蠕虫的寿命。 在具体目标 III 中，我们将 检查热量限制和碳水化合物诱导的效果 高胰岛素血症对 AMP 激酶和 DAF-16 同系物活性的影响