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First published online 25 November 2008
doi: 10.1242/jcs.030098

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p53 is localized to a sub-nucleolar compartment after proteasomal inhibition in an energy-dependent manner

¹ Department of Biological Sciences, Columbia University, New York, NY 10027, USA
² Herbert Irving Comprehensive Cancer Center, Columbia University, New York, NY 10032, USA
³ Laboratoire de Biologie Moléculaire de la Cellule, UMR 5161, Ecole Normale Supérieure de Lyon 46, Allée d'Italie, 69364 Lyon, Cedex 07, France

View larger version (32K): [in this window] [in a new window]   Fig. 1. p53 localizes to the FC of the nucleolus after MG132 treatment. (A) Schematic diagram of the nucleolus with the fibrillar center (FC) in the middle, surrounded by the dense fibrillar center (DFC) and the granular components (GCs). Some nucleoli can have several FCs. (B-D) H1299 cells were transfected with wild-type p53 and after 24 hours were treated with different doses (5 µM, 10 µM and 20 µM for 6-10 hours and 1 µM, 5 µM and 10 µM for 24 hours) of MG132 for various amount of time, as indicated. Western blotting of p53 was performed using PAb DO-1 for p53, anti-nucleolin and anti-actin antibodies (B). Transfected H1299 cells as in B were fixed with 4% paraformaldehyde and then examined by immunofluorescence analysis. The cells were visualized by confocal laser scanning and DIC microscopy (C). The percentage of cells with accumulated nucleolar p53 was estimated by counting at least 200 cells in two separate experiments (D). (E) H1299 cells were transfected, treated and analyzed as describe above and immunofluorescence was performed using PAb DO-1 and PAb 1801 for p53 and anti-UBF antibodies.

View larger version (32K): [in this window] [in a new window]   Fig. 2. p53 fused to the HIV-1 Rev nucleolar localization signal sequence associates with the DFC and this localization is independent of treatment with MG132. (A) Diagram of the HIV-1 Rev NoLSC'p53 in which HIV-1 Rev NoLS was tagged to the carboxyl terminus of wild-type p53. (B) H1299 cells were transfected for 24 hours with either empty vector, or vectors expressing wild-type p53 or Rev NoLSC'p53, then cells were either left untreated or treated with MG132 (10 µM) for 8 hours, solubilized in SDS electrophoresis sample buffer followed by immunoblotting with PAb DO-1, anti-HSP70, anti-nucleolin or anti-actin antibodies as a loading control. (C) H1299 cells transfected as in B were fixed with 4% paraformaldehyde, and immunostaining and visualization were performed as described in Fig. 1C. (D) Bar chart showing the percentage of cells with nucleolar localization of wild-type p53 and Rev NoLSC'p53. The data represent two independent experiments in which at least 200 cells were counted.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Genotoxic stress eliminates wild-type p53, Rev NoLSC'p53 and Rev sub-nucleolar association but only wild-type p53 nucleolar localization was ATP dependent. (A) H1299 p53-null cells were transfected with 1 µg of the following plasmids: wild-type p53, Rev NoLSC'p53 and Rev-GFP, and were either left untreated or were treated with daunorubicin (D) for 16 hours. 8 hours before the cells were lysed they were treated with 10 µM MG132 (M). Alternatively, 45 minutes before the cells were lysed they were treated with azide (a) as indicated. Combined treatments with MG132 and azide (Ma) and MG132 and daunorubicin (MD) were also performed, as indicated. Cells were then lysed in electrophoresis sample buffer and were subjected to western blotting. (B) Parallel treatments as in A were used to perform immunofluorescence experiments as described in Fig. 1C and Materials and Methods. (C) To quantify the immunofluorescence results, approximately 200 cells were counted from different fields in two independent experiments, and the results are presented as the percentage of cells with accumulated p53 or Rev NoLSC'p53 in the FC of the nucleolus.

View larger version (14K): [in this window] [in a new window]   Fig. 4. MG132 stimulates p53-ubiquitin nucleolar localization. H1299 cells were transfected with p53 (A) or p53-ubiqAA (B) with or without MG132 treatment. Immunofluorescence was performed as previously described in Fig. 1C and Materials and Methods.

View larger version (79K): [in this window] [in a new window]   Fig. 5. Accumulation of p53 in the nucleolus in human bladder and lung carcinomas. Lung and bladder carcinoma tissues stained for p53 by immunohistochemistry (A) and co-stained for p53 and UBF by immunofluorescence (B). Immunohistochemistry and immunofluorescence were performed as described in Materials and Methods. Red arrows indicate cells with nucleolar accumulation of p53 and black arrows indicate no nucleolar association of p53.

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