The mechanism by which hypoxia induces gene transcription involves the inhibition of HIF-1α (hypoxia-inducible factor-1 α subunit) PHD (prolyl hydroxylase) activity, which prevents the VHL (von Hippel-Lindau)-dependent targeting of HIF-1α to the ubiquitin/proteasome pathway. HIF-1α thus accumulates and promotes gene transcription. In the present study, first we provide direct biochemical evidence for the presence of a conserved hypoxic signalling pathway in Drosophila melanogaster. An assay for 2-oxoglutarate-dependent dioxygenases was developed using Drosophila embryonic and larval homogenates as a source of enzyme. Drosophila PHD has a low substrate specificity and hydroxylates key proline residues in the ODD (oxygen-dependent degradation) domains of human HIF-1α and Similar, the Drosophila homologue of HIF-1α. The enzyme promotes human and Drosophila [35S]VHL binding to GST (glutathione S-transferase)–ODD-domain fusion protein. Hydroxylation is enhanced by proteasomal inhibitors and was ascertained using an anti-hydroxyproline antibody. Secondly, by using transgenic flies expressing a fusion protein that combined an ODD domain and the green fluorescent protein (ODD–GFP), we analysed the hypoxic cascade in different embryonic and larval tissues. Hypoxic accumulation of the reporter protein was observed in the whole tracheal tree, but not in the ectoderm. Hypoxic stabilization of ODD–GFP in the ectoderm was restored by inducing VHL expression in these cells. These results show that Drosophila tissues exhibit different sensitivities to hypoxia.
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Research Article|
December 23 2005
Analysis of the hypoxia-sensing pathway in Drosophila melanogaster
Nathalie Arquier;
Nathalie Arquier
1
*Neurobiologie Vasculaire, INSERM U615, Université de Nice Sophia-Antipolis, Parc Valrose, Nice 06108, Nice Cedex 02, France
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Paul Vigne;
Paul Vigne
1
*Neurobiologie Vasculaire, INSERM U615, Université de Nice Sophia-Antipolis, Parc Valrose, Nice 06108, Nice Cedex 02, France
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Eric Duplan;
Eric Duplan
*Neurobiologie Vasculaire, INSERM U615, Université de Nice Sophia-Antipolis, Parc Valrose, Nice 06108, Nice Cedex 02, France
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Tien Hsu;
Tien Hsu
†Department of Pathology and Laboratory Medicine, and Hollings Cancer Center, Medical University of South Carolina, 86 Jonathan Lucas St, Rm 330, Charleston, SC 29425, U.S.A.
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Pascal P. Therond;
Pascal P. Therond
‡Institut de Signalisation, Biologie du Développement et Cancer, CNRS UMR 6543, Université de Nice Sophia-Antipolis, Parc Valrose, Nice 06108, Nice Cedex 02, France
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Christian Frelin;
Christian Frelin
*Neurobiologie Vasculaire, INSERM U615, Université de Nice Sophia-Antipolis, Parc Valrose, Nice 06108, Nice Cedex 02, France
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Gisela D'Angelo
*Neurobiologie Vasculaire, INSERM U615, Université de Nice Sophia-Antipolis, Parc Valrose, Nice 06108, Nice Cedex 02, France
2To whom correspondence should be addressed (email dangelo@unice.fr).
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Publisher: Portland Press Ltd
Received:
April 26 2005
Revision Received:
September 20 2005
Accepted:
September 21 2005
Accepted Manuscript online:
September 21 2005
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 393 (2): 471–480.
Article history
Received:
April 26 2005
Revision Received:
September 20 2005
Accepted:
September 21 2005
Accepted Manuscript online:
September 21 2005
Citation
Nathalie Arquier, Paul Vigne, Eric Duplan, Tien Hsu, Pascal P. Therond, Christian Frelin, Gisela D'Angelo; Analysis of the hypoxia-sensing pathway in Drosophila melanogaster. Biochem J 15 January 2006; 393 (2): 471–480. doi: https://doi.org/10.1042/BJ20050675
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