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: 6923254
• 负责人: MARIA Carmalita ALEXANDER-BRIDGES
• 金额: $ 5.03万
• 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/6923254
• 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(申请人提供)：蠕虫(线虫)的遗传证据 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Dauer 阻止并显示出更长的寿命和对氧化应激的抵抗能力，这是由于 DAF-16不受阻碍的行动。因此，DAF-16已经被证明是 激活超氧化物歧化酶基因(SOD)。在哺乳动物中，限制卡路里 低胰岛素信号被证明可以通过以下方式延缓衰老 机制包括增加DNA修复能力和减少 氧化应激。在低糖和循环胰岛素存在的情况下 在转录水平上，DAF-16同源物似乎是转录活性的。几个 实验室已经表明，在没有胰岛素的情况下，哺乳动物的同系物 DAF-16、FKHR、FKHRL1和AFX的激活基因转录 控制细胞凋亡和糖异生，而胰岛素可以抑制这一作用。 我们的目标是阐明DAF-16类因子激活的机制 在没有胰岛素的情况下转录。 卡路里限制和低糖可激活AMP激酶。我们发现AMP 激活剂能拮抗胰岛素对人肝癌细胞DAF-16的影响。 此外，AMP激酶还可以直接磷酸化DAF-16。我们建议 确定线虫中AMP激酶对DAF-16的调控及其作用 其同系物FKHR在哺乳动物细胞中的作用，可以解释热量摄入的能力 限制以减缓老化过程。在HepG2细胞中，胰岛素通过 DAF-16上的AKT位点抑制DAF-16的活性。我们发现AKT网站 在DAF-16中携带重叠的AMP激酶位点。在此的具体目标1中 提案，我们将确定AMP激酶是否调节DAF-16的活性 直接通过改变其磷酸化或通过调节其他 PI3激酶信号通路的元件。我们将研究以下方面的影响 AMP激酶对1)体内外磷酸化DAF-16的影响2)14-3-3 胰岛素存在和不存在时与DAF-16的结合以及3) DAF-16与其他增加其活性的蛋白质相互作用 结合/转录活性。在第二个具体目标中，我们将确定是否 AMP激酶可拮抗胰岛素信号转导细胞DAF-16的作用。 优雅，并延长寿命中的蠕虫。在具体目标三中，我们将 检查卡路里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