title: A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion.
abstract: 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.
authors.affiliation: Division of Molecular Physiology, School of Life Sciences and Wellcome Trust Biocentre, Dundee University, Dundee DD1 5EH, Scotland, U.K.
authors.forename: David A
authors.initials: DA
authors.lastname: Pan
authors.forename: D Grahame
authors.initials: DG
authors.lastname: Hardie
mesh_terms: D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002474:Cell-Free System; D004305:Dose-Response Relationship, Drug; D004331:Drosophila melanogaster / Q000201:enzymology*; D016678:Genome; D006801:Humans; D000860:Hypoxia; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000737:chemistry*; D009840:Oligomycins / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010802:Phylogeny; D011233:Precipitin Tests; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D012313:RNA / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D015398:Signal Transduction; D013997:Time Factors
chemical_list: D009097:Multienzyme Complexes; D009840:Oligomycins; D010455:Peptides; D012330:RNA, Double-Stranded; D012313:RNA; D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D000103:Acetyl-CoA Carboxylase