title: Differential sensitivity of hypoxia inducible factor hydroxylation sites to hypoxia and hydroxylase inhibitors.
abstract: Hypoxia inducible factor (HIF) is regulated by dual pathways involving oxygen-dependent prolyl and asparaginyl hydroxylation of its α-subunits. Prolyl hydroxylation at two sites within a central degradation domain promotes association of HIF-α with the von Hippel-Lindau ubiquitin E3 ligase and destruction by the ubiquitin-proteasome pathways. Asparaginyl hydroxylation blocks the recruitment of p300/CBP co-activators to a C-terminal activation domain in HIF-α. These hydroxylations are catalyzed by members of the Fe(II) and 2-oxoglutarate (2-OG) oxygenase family. Activity of the enzymes is suppressed by hypoxia, increasing both the abundance and activity of the HIF transcriptional complex. We have used hydroxy residue-specific antibodies to compare and contrast the regulation of each site of prolyl hydroxylation (Pro(402), Pro(564)) with that of asparaginyl hydroxylation (Asn(803)) in human HIF-1α. Our findings reveal striking differences in the sensitivity of these hydroxylations to hypoxia and to different inhibitor types of 2-OG oxygenases. Hydroxylation at the three sites in endogenous human HIF-1α proteins was suppressed by hypoxia in the order Pro(402) > Pro(564) > Asn(803). In contrast to some predictions from in vitro studies, prolyl hydroxylation was substantially more sensitive than asparaginyl hydroxylation to inhibition by iron chelators and transition metal ions; studies of a range of different small molecule 2-OG analogues demonstrated the feasibility of selectively inhibiting either prolyl or asparaginyl hydroxylation within cells.
authors.affiliation: Nuffield Department of Clinical Medicine, Henry Wellcome Building for Molecular Physiology, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom.
authors.forename: Ya-Min
authors.initials: YM
authors.lastname: Tian
authors.forename: Kar Kheng
authors.initials: KK
authors.lastname: Yeoh
authors.forename: Myung Kyu
authors.initials: MK
authors.lastname: Lee
authors.forename: Tuula
authors.initials: T
authors.lastname: Eriksson
authors.forename: Benedikt M
authors.initials: BM
authors.lastname: Kessler
authors.forename: Holger B
authors.initials: HB
authors.lastname: Kramer
authors.forename: Mariola J
authors.initials: MJ
authors.lastname: Edelmann
authors.forename: Carsten
authors.initials: C
authors.lastname: Willam
authors.forename: Christopher W
authors.initials: CW
authors.lastname: Pugh
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Schofield
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Ratcliffe
mesh_terms: D000818:Animals; D015687:Cell Hypoxia / Q000502:physiology; D004331:Drosophila melanogaster; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D056945:Hep G2 Cells; D006801:Humans; D006900:Hydroxylation / Q000502:physiology; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D020014:K562 Cells; D008297:Male; D010105:Oxygenases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D050880:p300-CBP Transcription Factors
chemical_list: D004791:Enzyme Inhibitors; C497442:HIF1A protein, human; C497444:Hif1a protein, rat; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010105:Oxygenases; D050880:p300-CBP Transcription Factors; D044767:Ubiquitin-Protein Ligases