Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress.
The tumor suppressor p53 is a nucleocytoplasmic shuttling protein that accumulates in the nucleus of cells exposed to various cellular stresses. One important role of nuclear p53 is to mobilize a stress response by transactivating target genes such as the p21(Waf1) gene. In this study, we investigated more closely the localization of p53 in cells following various stresses. Immunocytochemistry of fixed human fibroblasts treated with either UV light, the kinase and transcription inhibitor DRB or the proteasome inhibitor MG132 revealed abundant p53 localized to the nucleus. When cells treated with UV or DRB were permeabilized prior to fixation to allow soluble proteins to diffuse, the nuclear p53 signal was abolished. However, in cells treated with MG132, residual p53 localized to distinct large foci. Furthermore, nucleolin co-localized with p53 to these foci, suggesting that these foci were nucleolar structures. Interestingly, the MDM2 protein was found to co-localize with p53 to nucleolar structures following proteasome inhibition. Our results suggest that the p53 proteins accumulating in the nucleus following UV-irradiation or blockage of transcription are freely soluble and, thus, should be able to roam the nucleus to ensure high occupancy of p53 binding sites. However, inhibition of proteasome activity may be a unique stress in that it leads to the sequestering of p53 proteins to the nucleolus, thereby blunting the p53-mediated transactivation of target genes.
Klibanov
S A
SA
Dept of Radiation Oncology, Division of Cancer Biology, University of Michigan Comprehensive Cancer Center, Ann Arbor, MI, USA.
O'Hagan
H M
HM
Ljungman
M
M
D015152:Blotting, Northern; D002466:Cell Nucleolus / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000494:pharmacology; D004058:Diffusion; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D009687:Nuclear Proteins; D010750:Phosphoproteins / Q000032:analysis / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D012319:RNA Polymerase II / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000032:analysis; D016601:RNA-Binding Proteins / Q000032:analysis / Q000378:metabolism; D012995:Solubility; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000378:metabolism*; D014466:Ultraviolet Rays; D000097796:Nucleolin
D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D004004:Dichlororibofuranosylbenzimidazole; D007976:Leupeptins; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D010750:Phosphoproteins; D046988:Proteasome Endopeptidase Complex; D011518:Proto-Oncogene Proteins; D051736:Proto-Oncogene Proteins c-mdm2; D012319:RNA Polymerase II; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D016159:Tumor Suppressor Protein p53; D000097796:Nucleolin; C493956:CDKN1A protein, human; C497206:MDM2 protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde