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.
MeSH Terms
Amino Acid Sequence
Anaphase-Promoting Complex-Cyclosome
Animals
Carrier Proteins/metabolism,physiology
Cell Line, Tumor
Cysteine Endopeptidases/metabolism
DNA-Binding Proteins/metabolism,physiology
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
Neoplasm Proteins
Nuclear Proteins/metabolism,physiology
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,physiology
Transcription, Genetic
Transfection
Tryptophan/chemistry
Tumor Suppressor Protein p53/metabolism
Ubiquitin/metabolism
Ubiquitin-Conjugating Enzymes/chemistry
Ubiquitin-Protein Ligase Complexes/biosynthesis
Ubiquitin-Protein Ligases/metabolism,physiology
Chemicals
Carrier Proteins
DNA-Binding Proteins
Immediate-Early Proteins
Iron-Binding Proteins
Luminescent Proteins
Multienzyme Complexes
Neoplasm Proteins
Nuclear Proteins
Proto-Oncogene Proteins
Recombinant Proteins
Transcription Factors
Tumor Suppressor Protein p53
Ubiquitin
Green Fluorescent Proteins
Tryptophan
CDC34 protein, human
UBE2D1 protein, human
UBE2D3 protein, human
UBE2H protein, human
UBE2L3 protein, human
Ubiquitin-Conjugating Enzymes
Ubiquitin-Protein Ligase Complexes
Anaphase-Promoting Complex-Cyclosome
MDM2 protein, human
Proto-Oncogene Proteins c-mdm2
TOPORS protein, human
Ubiquitin-Protein Ligases
Vmw110 protein, Human herpesvirus 1
Glutathione Transferase
Cysteine Endopeptidases
Proteasome Endopeptidase Complex
Authors & Affiliations
10 authors, click to expand affiliations / ORCID
Rajendra Rajeev
Department of Pharmacology, The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08901, USA.
Malegaonkar Diptee
Pungaliya Pooja
Marshall Henderson
Rasheed Zeshaan
Brownell James
Liu Leroy F
Lutzker Stuart
Saleem Ahamed
Rubin Eric H
