Abstract
In normoxic cells the hypoxia-inducible factor-1 alpha (HIF-1 alpha) is rapidly degraded by the ubiquitin-proteasome pathway, and activation of HIF-1 alpha to a functional form requires protein stabilization. Here we show that the product of the von Hippel-Lindau (VHL) tumor suppressor gene mediated ubiquitylation and proteasomal degradation of HIF-1 alpha under normoxic conditions via interaction with the core of the oxygen-dependent degradation domain of HIF-1 alpha. The region of VHL mediating interaction with HIF-1 alpha overlapped with a putative macromolecular binding site observed within the crystal structure of VHL. This motif of VHL also represents a mutational hotspot in tumors, and one of these mutations impaired interaction with HIF-1 alpha and subsequent degradation. Interestingly, the VHL binding site within HIF-1 alpha overlapped with one of the minimal transactivation domains. Protection of HIF-1 alpha against degradation by VHL was a multistep mechanism, including hypoxia-induced nuclear translocation of HIF-1 alpha and an intranuclear hypoxia-dependent signal. VHL was not released from HIF-1 alpha during this process. Finally, stabilization of HIF-1 alpha protein levels per se did not totally bypass the need of the hypoxic signal for generating the transactivation response.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Binding Sites
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COS Cells
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Cell Line
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Crystallography, X-Ray
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Cysteine Endopeptidases / metabolism
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DNA-Binding Proteins / chemistry
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DNA-Binding Proteins / genetics
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DNA-Binding Proteins / metabolism*
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Fungal Proteins / metabolism
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Green Fluorescent Proteins
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Humans
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Hypoxia*
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Hypoxia-Inducible Factor 1
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Hypoxia-Inducible Factor 1, alpha Subunit
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Immunoblotting
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Ligases*
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Luminescent Proteins / metabolism
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Models, Biological
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Molecular Sequence Data
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Multienzyme Complexes / metabolism
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Mutation
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Nuclear Proteins / chemistry
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism*
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Oxygen / metabolism
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Plasmids / metabolism
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Precipitin Tests
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Proteasome Endopeptidase Complex
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Protein Structure, Tertiary
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Proteins / chemistry
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Proteins / genetics
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Proteins / metabolism*
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Saccharomyces cerevisiae Proteins*
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Sequence Homology, Amino Acid
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Signal Transduction
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Transcription Factors / metabolism
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Transcriptional Activation
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Transfection
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Tumor Suppressor Proteins*
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Ubiquitin-Protein Ligases*
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Ubiquitins / metabolism
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Von Hippel-Lindau Tumor Suppressor Protein
Substances
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DNA-Binding Proteins
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Fungal Proteins
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GAL4 protein, S cerevisiae
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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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Luminescent Proteins
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Multienzyme Complexes
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Nuclear Proteins
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Proteins
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Saccharomyces cerevisiae Proteins
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Transcription Factors
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Tumor Suppressor Proteins
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Ubiquitins
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Green Fluorescent Proteins
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Ubiquitin-Protein Ligases
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Von Hippel-Lindau Tumor Suppressor Protein
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex
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Ligases
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VHL protein, human
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Oxygen