HIF (hypoxia-inducible factor) is an αβ transcription factor that modulates the hypoxic response in many animals. The cellular abundance and activity of HIF-α are regulated by its post-translational hydroxylation. The hydroxylation of HIF is catalysed by PHD (prolyl hydroxylase domain) enzymes and FIH (factorinhibiting HIF), all of which are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. FIH hydroxylates a conserved asparagine residue in HIF-α (Asn-803), which blocks the binding of HIF to the transcriptional co-activator p300, preventing transcription of hypoxia-regulated genes under normoxic conditions. In the present paper, we report studies on possible mechanisms for the regulation of FIH activity. Recently solved crystal structures of FIH indicate that it is homodimeric. Site-directed mutants of FIH at residues Leu-340 and Ile-344, designed to disrupt dimerization, were generated in order to examine the importance of the dimeric state in determining FIH activity. A single point mutant, L340R (Leu-340→Arg), was shown to be predominantly monomeric and to have lost catalytic activity as measured by assays monitoring 2-oxoglutarate turnover and asparagine hydroxylation. In contrast, the I344R (Ile-344→Arg) mutant was predominantly dimeric and catalytically active. The results imply that the homodimeric form of FIH is required for productive substrate binding. The structural data also revealed a hydrophobic interaction formed between FIH and a conserved leucine residue (Leu-795) on the HIF substrate, which is close to the dimer interface. A recent report has revealed that phosphorylation of Thr-796, which is adjacent to Leu-795, enhances the transcriptional response in hypoxia. Consistent with this, we show that phosphorylation of Thr-796 prevents the hydroxylation of Asn-803 by FIH.
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Research Article|
October 26 2004
Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity
David E. LANCASTER;
David E. LANCASTER
1
*Department of Chemistry and the Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, U.K.
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Luke A. McNEILL;
*Department of Chemistry and the Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, U.K.
2To whom correspondence should be addressed (email christopher.schofield@chem.ox.ac.uk or luke.mcneill@chem.ox.ac.uk).
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Michael A. McDONOUGH;
Michael A. McDONOUGH
*Department of Chemistry and the Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, U.K.
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Robin T. APLIN;
Robin T. APLIN
*Department of Chemistry and the Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, U.K.
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Kirsty S. HEWITSON;
Kirsty S. HEWITSON
*Department of Chemistry and the Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, U.K.
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Christopher W. PUGH;
Christopher W. PUGH
†The Henry Wellcome Building of Genomic Medicine, Roosevelt Drive, Oxford OX3 7BN, U.K.
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Peter J. RATCLIFFE;
Peter J. RATCLIFFE
†The Henry Wellcome Building of Genomic Medicine, Roosevelt Drive, Oxford OX3 7BN, U.K.
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Christopher J. SCHOFIELD
Christopher J. SCHOFIELD
2
*Department of Chemistry and the Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, Mansfield Road, Oxford OX1 3TA, U.K.
2To whom correspondence should be addressed (email christopher.schofield@chem.ox.ac.uk or luke.mcneill@chem.ox.ac.uk).
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Publisher: Portland Press Ltd
Received:
May 04 2004
Revision Received:
June 22 2004
Accepted:
July 08 2004
Accepted Manuscript online:
July 08 2004
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2004
Biochem J (2004) 383 (3): 429–437.
Article history
Received:
May 04 2004
Revision Received:
June 22 2004
Accepted:
July 08 2004
Accepted Manuscript online:
July 08 2004
Citation
David E. LANCASTER, Luke A. McNEILL, Michael A. McDONOUGH, Robin T. APLIN, Kirsty S. HEWITSON, Christopher W. PUGH, Peter J. RATCLIFFE, Christopher J. SCHOFIELD; Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity. Biochem J 1 November 2004; 383 (3): 429–437. doi: https://doi.org/10.1042/BJ20040735
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