Abstract
The human topoisomerase I- and p53-binding protein topors contains a highly conserved, N-terminal C3HC4-type RING domain that is homologous to the RING domains of known E3 ubiquitin ligases. We demonstrate that topors functions in vitro as a RING-dependent E3 ubiquitin ligase with the E2 enzymes UbcH5a, UbcH5c, and UbcH6 but not with UbcH7, CDC34, or UbcH2b. Additional studies indicate that a conserved tryptophan within the topors RING domain is required for ubiquitination activity. Furthermore, both in vitro and cellular studies implicate p53 as a ubiquitination substrate for topors. Similar to MDM2, overexpression of topors results in a proteasome-dependent decrease in p53 protein expression in a human osteosarcoma cell line. These results are similar to the recent finding that a Drosophila topors orthologue ubiquitinates the Hairy transcriptional repressor and suggest that topors functions as a ubiquitin ligase for multiple transcription factors.
Publication types
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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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Anaphase-Promoting Complex-Cyclosome
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Animals
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Carrier Proteins / metabolism
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Carrier Proteins / physiology*
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Cell Line, Tumor
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Cysteine Endopeptidases / metabolism
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DNA-Binding Proteins / metabolism
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DNA-Binding Proteins / physiology*
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Drosophila
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Electrophoresis, Polyacrylamide Gel
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Glutathione Transferase / metabolism
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Green Fluorescent Proteins
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Humans
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Immediate-Early Proteins / metabolism
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Immunoblotting
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Iron-Binding Proteins / chemistry
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Luminescent Proteins / metabolism
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Mass Spectrometry
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Molecular Sequence Data
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Multienzyme Complexes / metabolism
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Neoplasm Proteins*
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Nuclear Proteins / metabolism
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Nuclear Proteins / physiology*
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Plasmids / metabolism
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Proteasome Endopeptidase Complex
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Protein Structure, Tertiary
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-mdm2
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Recombinant Proteins / chemistry
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Sequence Homology, Amino Acid
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Silver Staining
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Transcription Factors / metabolism
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Transcription Factors / physiology*
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Transcription, Genetic
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Transfection
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Tryptophan / chemistry
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Tumor Suppressor Protein p53 / metabolism*
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Ubiquitin / metabolism*
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Ubiquitin-Conjugating Enzymes / chemistry
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Ubiquitin-Protein Ligase Complexes / biosynthesis
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Ubiquitin-Protein Ligases / metabolism
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Ubiquitin-Protein Ligases / physiology*
Substances
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Carrier Proteins
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DNA-Binding Proteins
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Immediate-Early Proteins
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Iron-Binding Proteins
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Luminescent Proteins
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Multienzyme Complexes
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Neoplasm Proteins
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Nuclear Proteins
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Proto-Oncogene Proteins
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Recombinant Proteins
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Transcription Factors
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Tumor Suppressor Protein p53
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Ubiquitin
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Green Fluorescent Proteins
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Tryptophan
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CDC34 protein, human
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UBE2D1 protein, human
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UBE2D3 protein, human
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UBE2H protein, human
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UBE2L3 protein, human
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Ubiquitin-Conjugating Enzymes
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Ubiquitin-Protein Ligase Complexes
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Anaphase-Promoting Complex-Cyclosome
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MDM2 protein, human
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Proto-Oncogene Proteins c-mdm2
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TOPORS protein, human
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Ubiquitin-Protein Ligases
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Vmw110 protein, Human herpesvirus 1
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Glutathione Transferase
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Cysteine Endopeptidases
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Proteasome Endopeptidase Complex