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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.

SUBMITTER: Pan DA

PROVIDER: S-EPMC1222868 | biostudies-other | 2002 Oct

REPOSITORIES: biostudies-other

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Project description:Living cells obtain energy either by oxidizing reduced compounds of organic or mineral origin or by absorbing light. Whichever energy source is used, some of the energy released is conserved by converting adenosine diphosphate (ADP) to adenosine triphosphate (ATP), which are analogous to the chemicals in a rechargeable battery. The energy released by the conversion of ATP back to ADP is used to drive most energy-requiring processes, including cell growth, cell division, communication and movement. It is clearly essential to life that the production and consumption of ATP are always maintained in balance, and the AMP-activated protein kinase (AMPK) is one of the key cellular regulatory systems that ensures this. In eukaryotic cells (cells with nuclei and other internal membrane-bound structures, including human cells), most ATP is produced in mitochondria, which are thought to have been derived by the engulfment of oxidative bacteria by a host cell not previously able to use molecular oxygen. AMPK is activated by increasing AMP or ADP (AMP being generated from ADP whenever ADP rises) coupled with falling ATP. Relatives of AMPK are found in essentially all eukaryotes, and it may have evolved to allow the host cell to monitor the output of the newly acquired mitochondria and step their ATP production up or down according to the demand. Structural studies have illuminated how AMPK achieves the task of detecting small changes in AMP and ADP, despite the presence of much higher concentrations of ATP. Recently, it has been shown that AMPK can also sense the availability of glucose, the primary carbon source for most eukaryotic cells, via a mechanism independent of changes in AMP or ADP. Once activated by energy imbalance or glucose lack, AMPK modifies many target proteins by transferring phosphate groups to them from ATP. By this means, numerous ATP-producing processes are switched on (including the production of new mitochondria) and ATP-consuming processes are switched off, thus restoring energy homeostasis. Drugs that modulate AMPK have great potential in the treatment of metabolic disorders such as obesity and Type 2 diabetes, and even cancer. Indeed, some existing drugs such as metformin and aspirin, which were derived from traditional herbal remedies, appear to work, in part, by activating AMPK.

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A homologue of AMP-activated protein kinase in Drosophila melanogaster is sensitive to AMP and is activated by ATP depletion.

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