title: UBE2Q1 in a Human Breast Carcinoma Cell Line: Overexpression and Interaction with p53.
abstract: The p53 tumor suppressor protein is a principal mediator of growth arrest, senescence, and apoptosis in response to a broad array of cellular damage. p53 is a substrate for the ubiquitin-proteasome system, however, the ubiquitin-conjugating enzymes (E2s) involved in p53 ubiquitination have not been well studied. UBE2Q1 is a novel E2 ubiquitin conjugating enzyme gene. Here, we investigated the effect of UBE2Q1 overexpression on the level of p53 in the MDA-MB-468 breast cancer cell line as well as the interaction between UBE2Q1 and p53. By using a lipofection method, the p53 mutated breast cancer cell line, MDA-MB-468, was transfected with the vector pCMV6-AN-GFP, containing UBE2Q1 ORF. Western blot analysis was employed to verify the overexpression of UBE2Q1 in MDA-MB-468 cells and to evaluate the expression level of p53 before and after cell transfection. Immunoprecipitation and GST pull-down protocols were used to investigate the binding of UBE2Q1 to p53. We established MDA-MB-468 cells that transiently expressed a GFP fusion proteins containing UBE2Q1 (GFP-UBE2Q1). Western blot analysis revealed that levels of p53 were markedly lower in UBE2Q1 transfected MDA-MB-468 cells as compared with control MDA-MB-468 cells. Both in vivo and in vitro data showed that UBE2Q1 co-precipitated with p53 protein. Our data for the first time showed that overexpression of UBE2Q1can lead to the repression of p53 in MDA-MB-468 cells. This repression of p53 may be due to its UBE2Q1 mediated ubiquitination and subsequent proteasome degradation, a process that may involve direct interaction of UBE2Q1with p53.
authors.affiliation: Departments of Biochemistry- Recombinant Protein Laboratory, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran E-mail : owjiaa@sums.ac.ir.
authors.forename: Sayed Mohammad
authors.initials: SM
authors.lastname: Shafiee
authors.forename: Mozhgan
authors.initials: M
authors.lastname: Rasti
authors.forename: Atefeh
authors.initials: A
authors.lastname: Seghatoleslam
authors.forename: Tayebeh
authors.initials: T
authors.lastname: Azimi
authors.forename: Ali Akbar
authors.initials: AA
authors.lastname: Owji
mesh_terms: D015153:Blotting, Western; D001943:Breast Neoplasms / Q000378:metabolism*; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D009154:Mutation / Q000235:genetics; D054730:Protein Interaction Domains and Motifs; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism*
chemical_list: C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53; C581814:UBE2Q1 protein, human; D044763:Ubiquitin-Conjugating Enzymes