Pan David A, Hardie D Grahame
Division of Molecular Physiology, School of Life Sciences and Wellcome Trust Biocentre, Dundee University, Dundee DD1 5EH, Scotland, U.K.
Biochem J. 2002 Oct 1;367(Pt 1):179-86. doi: 10.1042/BJ20020703.
We have identified single genes encoding homologues of the alpha, beta and gamma subunits of mammalian AMP-activated protein kinase (AMPK) in the genome of Drosophila melanogaster. Kinase activity could be detected in extracts of a Drosophila cell line using the SAMS peptide, which is a relatively specific substrate for the AMPK/SNF1 kinases in mammals and yeast. Expression of double stranded (ds) RNAs targeted at any of the putative alpha, beta or gamma subunits ablated this activity, and abolished expression of the alpha subunit. The Drosophila kinase (DmAMPK) was activated by AMP in cell-free assays (albeit to a smaller extent than mammalian AMPK), and by stresses that deplete ATP (oligomycin and hypoxia), as well as by carbohydrate deprivation, in intact cells. Using a phosphospecific antibody, we showed that activation was associated with phosphorylation of a threonine residue (Thr-184) within the 'activation loop' of the alpha subunit. We also identified a homologue of acetyl-CoA carboxylase (DmACC) in Drosophila and, using a phosphospecific antibody, showed that the site corresponding to the regulatory AMPK site on the mammalian enzyme became phosphorylated in response to oligomycin or hypoxia. By immunofluorescence microscopy of oligomycin-treated Dmel2 cells using the phosphospecific antibody, the phosphorylated DmAMPK alpha subunit was mainly detected in the nucleus. Our results show that the AMPK system is highly conserved between insects and mammals. Drosophila cells now represent an attractive system to study this pathway, because of the small, well-defined genome and the ability to ablate expression of specific gene products using interfering dsRNAs.
我们在黑腹果蝇基因组中鉴定出了编码哺乳动物AMP活化蛋白激酶（AMPK）α、β和γ亚基同源物的单个基因。使用SAMS肽可在果蝇细胞系提取物中检测到激酶活性，SAMS肽是哺乳动物和酵母中AMPK/SNF1激酶相对特异的底物。针对任何一个假定的α、β或γ亚基的双链（ds）RNA的表达消除了这种活性，并消除了α亚基的表达。在无细胞试验中，果蝇激酶（DmAMPK）被AMP激活（尽管程度比哺乳动物AMPK小），在完整细胞中被消耗ATP的应激（寡霉素和缺氧）以及碳水化合物剥夺激活。使用磷酸特异性抗体，我们表明激活与α亚基“激活环”内的苏氨酸残基（Thr-184）磷酸化有关。我们还在果蝇中鉴定出了乙酰辅酶A羧化酶（DmACC）的同源物，并使用磷酸特异性抗体表明，与哺乳动物酶上的调节性AMPK位点相对应的位点在寡霉素或缺氧刺激下发生了磷酸化。通过使用磷酸特异性抗体对寡霉素处理的Dmel2细胞进行免疫荧光显微镜观察，磷酸化的DmAMPKα亚基主要在细胞核中被检测到。我们的结果表明，AMPK系统在昆虫和哺乳动物之间高度保守。由于基因组小且定义明确，以及能够使用干扰性dsRNAs消除特定基因产物的表达，果蝇细胞现在是研究该途径的一个有吸引力的系统。
