Abstract
Hypoxia-inducible factor-1alpha (HIF-1alpha) is a global transcriptional regulator of the hypoxic response. Under normoxic conditions, HIF-1alpha is recognized by the von Hippel-Lindau tumor-suppressor protein (VHL), a component of an E3 ubiquitin ligase complex. This interaction thereby promotes the rapid degradation of HIF-1alpha. Under hypoxic conditions, HIF-1alpha is stabilized. We have previously shown that VHL binds in a hypoxia-sensitive manner to a 27-aa segment of HIF-1alpha, and that this regulation depends on a posttranslational modification of HIF-1alpha. Through a combination of in vivo coimmunoprecipitation assays using VHL and a panel of point mutants of HIF-1alpha in this region, as well as MS and in vitro binding assays, we now provide evidence that this modification, which occurs under normoxic conditions, is hydroxylation of Pro-564 of HIF-1alpha. The data furthermore show that this proline hydroxylation is the primary regulator of VHL binding.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Animals
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COS Cells
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Cell Hypoxia
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Chlorocebus aethiops
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Cobalt / pharmacology
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DNA-Binding Proteins / metabolism*
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HeLa Cells
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Humans
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Hydroxylation
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Hydroxyproline / chemistry
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Hydroxyproline / physiology*
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Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factor 1, alpha Subunit
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Ligases*
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Macromolecular Substances
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Molecular Sequence Data
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Nuclear Proteins / metabolism*
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Peptide Fragments / metabolism
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Point Mutation
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Proline / chemistry
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Protein Binding / drug effects
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Protein Processing, Post-Translational*
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Proteins / genetics
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Proteins / metabolism*
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Transcription Factors*
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Transfection
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Tumor Suppressor Proteins*
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Ubiquitin-Protein Ligases*
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Von Hippel-Lindau Tumor Suppressor Protein
Substances
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DNA-Binding Proteins
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HIF1A protein, human
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Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factor 1, alpha Subunit
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Macromolecular Substances
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Nuclear Proteins
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Peptide Fragments
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Proteins
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Recombinant Fusion Proteins
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Transcription Factors
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Tumor Suppressor Proteins
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Cobalt
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Proline
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Ubiquitin-Protein Ligases
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Von Hippel-Lindau Tumor Suppressor Protein
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Ligases
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VHL protein, human
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Hydroxyproline