[Nuclear import of p53 in relation to MDM2-mediated degradation and ubiquitination].
OBJECTIVE
To study the function of p53 nuclear import in murine double minute 2 (MDM2)-mediated ubiquitination and degradation.


METHODS
Plasmid containing mutant p53-GFP was constructed by site-directed mutagenesis by which 5 amino scid residues in the nuclear localization signal (NLS) were replaced by alanine to produce mutant p53KRKKK-GFP. After being fused with pEGF-Nuc (NLS containing SV40) to produce p53KRKKK-NLS-GFP, it was transfected into U20S cells. Localization, degradation and ubiquitination of p53 and MDM2 proteins were assessed by fluorescent staining, Western blot and ubiquitination analysis in MDM2 or MDM2-NLS co-transfected U20S cells.


RESULTS
p53KRKKK-GFP was located in cytoplasm, and was not degraded by either MDM2 or MDM2-NLS mutation, but could be ubiquitinated; p53KRKKK-NLS-GFP could be brought back to nucleus by SV-40 NLS, so could be both degraded and ubiquitinated by either MDM2 or MDM2-NLS; Wild type p53 and mutant NLS could be ubiquitinated by either wild type MDM2 or mutant NLS. Ubiquitination happened to be even more efficient in cytoplasm when p53KRKKK and MDM2-NLS co-localization, but not degraded.


CONCLUSIONS
Nuclear import is required for p53 degradation mediated by MDM2, but not for ubiquitination. p53 can be efficiently ubiquitinated in cytoplasm.
Li
Hui-ping
HP
Department of Oncology, Third Hospital, Peking University, Beijing 100083. huiping_l@hotmail.com
Zhang
Yan-ping
YP
D001859:Bone Neoplasms / Q000473:pathology; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism*; D012516:Osteosarcoma / Q000473:pathology; D010957:Plasmids; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D011994:Recombinant Proteins / Q000378:metabolism; D014162:Transfection; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
D019913:Nuclear Localization Signals; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2