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）：蠕虫中的遗传证据（秀丽隐杆线虫） 已经表明，胰岛素样分子通过PI3激酶和AKT/蛋白质作用 激酶B抑制叉子（FKH）转录因子的功能 DAF-16。 功能降低的胰岛素信号突变体发生DAUER 逮捕并显示出增加的寿命和抵抗力，抗氧化应激 DAF-16的不受阻碍的行动。 因此，DAF-16已显示 激活超氧化物歧化酶基因（SOD）。 在哺乳动物中，热量限制 并且已经显示低胰岛素信号传导会减慢衰老的速度 包括增加DNA修复能力和降低的机制 氧化应激。 在低葡萄糖和循环胰岛素的情况下 水平，DAF-16同源物似乎具有转录活性。 一些 实验室表明，在没有胰岛素的情况下，哺乳动物同源物 DAF-16，FKHR，FKHRL1和AFX激活基因的转录 控制凋亡，糖异生和胰岛素可以抑制这种作用。 我们的目标是阐明DAF-16类似因素激活的机制 在没有胰岛素的情况下转录。 热量限制和低葡萄糖会激活AMP激酶。 我们发现那个放大器 激酶可以防止胰岛素对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激酶对DAF-16的调节和调节。 其同源物在哺乳动物细胞中的同源物可以解释热量的能力 限制减慢老化过程。 在HEPG2细胞中，胰岛素信号传导通过 DAF-16中的AKT位点抑制DAF-16活性。 我们发现AKT网站 在DAF-16中，携带重叠的AMP激酶位点。 在其中的特定目标1中 提案，我们将确定AMP激酶是否调节DAF-16活性 直接通过调节其他人的磷酸化或间接改变其磷酸化 PI3激酶信号途径的元素。 我们将研究 AMP激酶在1）体外和体内的磷DAF-16磷酸化，2）14-3-3 在存在和不存在胰岛素的情况下与DAF-16结合，3） DAF-16与其他蛋白质的相互作用 结合/转录活性。 在特定的目标II中，我们将确定是否 AMP激酶可以抵消C中胰岛素信号传导的作用。 秀丽隐杆线，并在蠕虫中延长寿命。 在特定的目标III中，我们将 检查热量限制和碳水化合物诱导的效果 关于AMP激酶和DAF-16同源物活性的高胰岛素血症