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Type: Journal article
Title: Substrate requirements of the oxygen-sensing asparaginyl hydroxylase factor-inhibiting hypoxia-inducible factor
Author: Karttunen, S.
Stojkoski, C.
Kewley, R.
Booker, G.
Whitelaw, M.
Peet, D.
Citation: Biological Chemistry: official scientific journal of the GBM, 2004; 279(14):14391-14397
Publisher: Walter de Gruyter & Co
Issue Date: 2004
ISSN: 1431-6730
Organisation: Centre for the Molecular Genetics of Development
Statement of
Sarah Linke, Cvetan Stojkoski, Robyn J. Kewley, Grant W. Booker, Murray L. Whitelaw and Daniel J. Peet
Abstract: The hypoxia-inducible factor subunits 1 and 2 (HIF-1 and HIF-2) are subjected to oxygen-dependent asparaginyl hydroxylation, a modification that represses the carboxyl-terminal transactivation domain (CAD) at normoxia by preventing recruitment of the p300/cAMP-response element-binding protein coactivators. This hydroxylation is performed by the novel asparaginyl hydroxylase, factor-inhibiting HIF-1' (FIH-1), of which HIF-1 and HIF-2 are the only reported substrates. Here we investigated the substrate requirements of FIH-1 by characterizing its subcellular localization and by examining amino acids within the HIF-1 substrate for their importance in recognition and catalysis by FIH-1. Using immunohistochemistry, we showed that both endogenous and transfected FIH-1 are primarily confined to the cytoplasm and remain there under normoxia and following treatment with the hypoxia mimetic, dipyridyl. Individual alanine mutations of seven conserved amino acids flanking the hydroxylated asparagine in HIF-1 revealed the importance of the valine (Val-802) adjacent to the targeted asparagine. The HIF-1 CAD V802A mutant exhibited a 4-fold lower Vmax in enzyme assays, whereas all other mutants were hydroxylated as efficiently as the wild type HIF-1 CAD. Furthermore, in cell-based assays the transcriptional activity of V802A was constitutive, suggesting negligible normoxic hydroxylation in HEK293T cells, whereas the wild type and other mutants were repressed under normoxia. Molecular modeling of the HIF-1 CAD V802A in complex with FIH-1 predicted an alteration in asparagine positioning compared with the wild type HIF-1 CAD, providing an explanation for the impaired catalysis observed and confirming the importance of Val-802 in asparaginyl hydroxylation by FIH-1.
Keywords: Kidney
Cell Line
Mixed Function Oxygenases
DNA-Binding Proteins
Receptors, Aryl Hydrocarbon
Transcription Factors
Repressor Proteins
Crystallography, X-Ray
Amino Acid Sequence
Protein Structure, Tertiary
Substrate Specificity
Molecular Sequence Data
Aryl Hydrocarbon Receptor Nuclear Translocator
Hypoxia-Inducible Factor 1, alpha Subunit
Description: Copyright © 2004 by The American Society for Biochemistry and Molecular Biology
DOI: 10.1074/jbc.M313614200
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Centre for the Molecular Genetics of Development publications
Molecular and Biomedical Science publications

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