Topors Functions as an E3 Ubiquitin Ligase with Specific E2 Enzymes and Ubiquitinates p53

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.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, a p53 and topoisomerase I-binding RING finger protein, is a coactivator of p53 in growth suppression induced by DNA damage

“…Therefore, topors could affect the stability of p53 via covalent modification of p53 by ubiquitin isopeptide. This observation, however, is not necessarily consistent with our and others' previous observations suggesting topors as a likely tumor suppressor protein since polyubiquitination of p53 mediated by GFPtopors induces proteasome-dependent downregulation of p53 in U2-OS cells (Chu et al, 2001;Rajendra et al, 2004;Saleem et al, 2004). This implies that regulation of p53 by topors may involve not only ubiquitination but also some other molecular mechanism.…”

Stimulation of in vitro sumoylation by Slx5–Slx8: Evidence for a functional interaction with the SUMO pathway

“…Precedent for such a dual-activity protein is provided by the conserved RING-finger protein, Topors. Topors binds DNA topoisomerase I [50] and p53 [51,52] and it displays both Ub E3 ligase [53,54] and SUMO ligase activity [55]. Topors Ub ligase activity is dependent on its RING-finger domain, while its SUMO ligase activity is not [55].…”

E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1)

“…RING finger E3 ligases have been reported to catalyze autoubiquitinylation (15) and substrate ubiquitinylation (34), as well as the synthesis of unanchored polyubiquitin chains in vitro (23,24). Although previously unrecognized substrate(s) for many proteins containing RING finger domain(s) continue to be identified, for the majority, including ARD1, the physiological substrates remain unknown.…”