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.Topors was originally discovered in a screen for proteins that bind to the N terminus of topoisomerase I (1) and was also identified in a screen for proteins that interact with p53 (denoted p53BP3) (2). Furthermore, topors was identified in an assay for RING domain proteins that are expressed in normal lung tissue (denoted LUN) (3). While topors is widely expressed in normal human tissues, topors mRNA and protein levels are commonly decreased or undetectable in colon adenocarcinomas and cell lines (4). The topors protein contains an N-terminal C3HC4-type RING domain that is conserved in orthologues from various species (5) and is closely related in sequence to the RING domains of known E3 ubiquitin ligases such as the herpesvirus protein ICP0 and Cbl (6, 7). Recently, a Drosophila topors orthologue was shown to interact physically and genetically with the Hairy transcriptional repressor (8). Furthermore, the Drosophila topors protein was shown to ubiquitinate Hairy in vitro and to decrease expression of an epitope-tagged Hairy protein in co-transfection studies (8).Topors is also known to associate with promyelocytic leukemia (PML) 1 nuclear bodies in the nuclei of exponentially growing cells (5, 9). Treatment with transcriptional inhibitors or with the topoisomerase I-targeting drug camptothecin results in rapid dispersion of topors to the nucleoplasm, suggesting that topors is involved in the cellular response to transcriptional perturbation (5).To gain insight into the role of the topors and the conserved RING domain, we determined whether topors functions as a ubiquitin ligase. Our results indicate that topors acts as a RING domain-dependent, E3 ubiquitin ligase with specific E2 enzymes. Similar to the E3 ubiquitin ligase Cbl, a conserved tryptophan within the topors RING domain is required for ubiquitin ligase activity. Furthermore, additional in vitro and cellular studies implicate p53 as a ubiquitination substrate for topors. EXPERIMENTAL PROCEDURESExpression Plasmids-A plasmid expressing a GST-N-terminal topors fusion protein (pGEX-topors) was constructed using...

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.

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