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https://hdl.handle.net/2440/3117
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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 1083-351X |
Organisation: | Centre for the Molecular Genetics of Development |
Statement of Responsibility: | 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 Humans Oxygen Mixed Function Oxygenases Asparagine DNA-Binding Proteins Receptors, Aryl Hydrocarbon Transcription Factors Repressor Proteins Crystallography, X-Ray Amino Acid Sequence Protein Structure, Tertiary Substrate Specificity Hydroxylation 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 |
Published version: | http://www.jbc.org/cgi/content/abstract/279/14/14391 |
Appears in Collections: | Aurora harvest 6 Centre for the Molecular Genetics of Development publications Molecular and Biomedical Science publications |
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