ABSTRACT
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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.
Subject(s)
Carrier Proteins/physiology , DNA-Binding Proteins/physiology , Neoplasm Proteins , Nuclear Proteins/physiology , Transcription Factors/physiology , Tumor Suppressor Protein p53/metabolism , Ubiquitin-Protein Ligases/physiology , Ubiquitin/metabolism , Amino Acid Sequence , Anaphase-Promoting Complex-Cyclosome , Animals , Carrier Proteins/metabolism , Cell Line, Tumor , Cysteine Endopeptidases/metabolism , DNA-Binding Proteins/metabolism , Drosophila , Electrophoresis, Polyacrylamide Gel , Glutathione Transferase/metabolism , Green Fluorescent Proteins , Humans , Immediate-Early Proteins/metabolism , Immunoblotting , Iron-Binding Proteins/chemistry , Luminescent Proteins/metabolism , Mass Spectrometry , Molecular Sequence Data , Multienzyme Complexes/metabolism , Nuclear Proteins/metabolism , Plasmids/metabolism , Proteasome Endopeptidase Complex , Protein Structure, Tertiary , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Silver Staining , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Transcription Factors/metabolism , Transcription, Genetic , Transfection , Tryptophan/chemistry , Ubiquitin-Conjugating Enzymes/chemistry , Ubiquitin-Protein Ligase Complexes/biosynthesis , Ubiquitin-Protein Ligases/metabolismABSTRACT
Topors was identified recently as a human protein that binds to topoisomerase I and p53. Topors contains a highly conserved RING domain and localizes in promyelocytic leukemia nuclear bodies. Relatively little is known regarding topors expression patterns or function. We now demonstrate that topors mRNA and protein are widely expressed in normal human tissues. By contrast, topors mRNA and protein levels are decreased or undetectable in colon adenocarcinomas relative to normal colon tissue, and expression of the topors protein is not detectable in several colon cancer cell lines. The human TOPORS gene is located on chromosome 9p21, with loss of heterozygosity in this region frequently observed in several different malignancies. While we were unable to detect loss of heterozygosity of the TOPORS gene in 16 sporadic colon cancer cases, increased methylation of a CpG island in the TOPORS promoter was evident in colon adenocarcinoma specimens relative to matched normal tissues. Additional studies indicate that forced expression of topors inhibits cellular proliferation and is associated with an accumulation of cells in the G(0)/G(1) phase of the cell cycle. This effect is independent of the topors RING domain and maps to a C-terminal region of the protein. These results suggest that topors functions as a negative regulator of cell growth, and possibly as a tumor suppressor.