doi: 10.1128/MCB.18.12.7288.
Nuclear export is required for degradation of endogenous p53 by MDM2 and human papillomavirus E6
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- PMID:9819415
- PMCID: PMC109310
- DOI: 10.1128/MCB.18.12.7288
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Nuclear export is required for degradation of endogenous p53 by MDM2 and human papillomavirus E6
D A Freedman et al. Mol Cell Biol.1998 Dec.
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Abstract
The MDM2 oncoprotein targets the p53 tumor suppressor protein for degradation when the two proteins are expressed in cells. The regulation of p53 levels by MDM2 requires the ability of MDM2 to be exported from the nucleus by utilizing its nuclear export signal (NES). The drug leptomycin B (LMB) blocks the formation of nuclear export complexes consisting of CRM1, RanGTP, and NES-containing proteins. It is predicted that LMB should inhibit nuclear-cytoplasmic shuttling by MDM2 and subsequently stabilize p53. This communication demonstrates that LMB treatment of various cell lines led to an increase in the steady-state levels of the p53 protein as a result of an increase in its stability. The stabilized p53 protein localized to the nucleus and was an active transcription factor. These results indicate that the low steady-state levels of p53 in the absence of DNA damage result from p53's nuclear export for cytoplasmic degradation. LMB also led to p53 stabilization in cell lines that contain human papillomavirus (HPV) DNA and express HPV E6, a protein that targets p53 for degradation. MDM2 is not necessary for E6-dependent degradation of p53, as evidenced by the observation that E6 promoted p53 degradation in cells lacking endogenous MDM2. In addition, LMB reduced E6's ability to degrade p53 in the absence of MDM2, demonstrating that complete degradation of p53 by E6 requires nuclear export and therefore likely occurs in cytoplasmic proteasomes. These data suggest that the nuclear export of p53 to the cytoplasm for degradation is a general mechanism for regulating p53 levels.
Figures
LMB increases p53 protein levels in several cell lines. LMB (5 ng/ml) was added 6 or 20 h prior to the harvest of the cells. The steady-state levels of p53, β-catenin, and Ran in each lysate were observed by separation of total protein by SDS-PAGE and immunoblotting with antibodies specific for each of the proteins. The name of each cell line is indicated at the top; less protein was used for the determination of p53 levels in SK-mel-2, due to the high levels of the mutant p53 protein in these cells. The addition of LMB led to an increase in the levels of the p53 protein in each cell line, except in the p53-null H1299 cell line. Such an effect of LMB is not seen in the levels of the β-catenin and Ran proteins, demonstrating the specificity of the observation for p53.
The p53 protein induced by LMB is localized to the nucleus. SJSA cells (A and B) and MCF-7 cells (C and D) were left untreated (A and C) or were treated with 5 ng of LMB per ml (B and D) 20 h prior to fixation. The p53 protein was detected with MAb 421, followed by goat anti-mouse IgG-Alexa568. In both cases, the induced p53 protein is localized to the nucleus of the cells following LMB treatment.
The p53 protein induced by LMB is transcriptionally active. Cells were treated with 5 ng of LMB per ml 6 and 20 h prior to harvesting. The steady-state levels of p53 and p21 in each lysate were observed by separation of total protein by SDS-PAGE followed by immunoblotting. MDM2 protein was immunoprecipitated prior to immunoblotting. The levels of MDM2 and p21 increased in response to LMB in wild-type p53-containing SJSA and MCF-7 cells, but not in SK-mel-2 and H1299 cells that lack functional p53 protein. A longer exposure is shown for the left half of the MDM2 blot because of the lower levels of MDM2 in these cells, and a shorter exposure is shown for the p21 blot of MCF-7 cells due to the higher levels of p21 in this cell line.
LMB leads to an increase in the half-life of p53 in 12(1) cells. (A) Five plates of 12(1) cells were pretreated with 5 ng of LMB per ml for 2.5 h prior to being prestarved for 30 min in media lacking methionine and then were labeled with 75 μCi of35S-Express per ml for 3 h, both in the presence of the same concentration of LMB. Six plates were prestarved and labeled similarly, but without the addition of LMB. Cell were washed, chased with media containing excess cold methionine with or without 5 ng of LMB per ml, and harvested at different times. Samples were immunoprecipitated with MAb 419 (419 IP) against simian virus 40 large T antigen as a negative control or with MAb 421 against p53, washed, and run on SDS-PAGE. Molecular weights (MW) are shown to the left (thousands). (B) The levels of p53 remaining at each time were quantified from four separate experiments on a phosphoimager, background was subtracted, and values were normalized to the samples that were not chased in the same experiment. The average fractions of p53 remaining and their associated standard deviations are shown at the various chase times on the left-hand plot, in the absence of LMB (solid line) and in the presence of 5 ng of LMB per ml (dashed line). The natural log (ln) transform of this plot is also shown on the right; linear regression of this plot was used to calculate the average half-life of p53 without (57 min) and with (236 min) the addition of 5 ng of LMB per ml. The difference between the fraction of p53 remaining at each time point in the presence or absence of LMB was tested by multivariate analysis of covariance with the Scheffé test for post hoc analysis. Asterisks (∗) denote time points that are significantly different between the two conditions (P < 0.01).
LMB induces the p53 protein in HPV E6-expressing cells. Cells were treated, lysates were prepared, and p53 was detected by immunoblotting as described in the legend to Fig. 1. Baculovirus-purified human p53 was loaded in the first lane as a positive control. HeLa cells contain HPV-18 sequences, while SiHa and CaSki cells contain HPV-16 sequences. The p53 protein levels in each of these cell lines increased after 20 h in the presence of 5 ng of LMB per ml. Molecular weights (MW) are shown to the left (thousands).
HPV E6 degradation of p53 is MDM2-independent and LMB sensitive. The 2KO cell line, which lacks endogenous MDM2, was transfected with pGL2-Control which leads to constitutive expression of luciferase (lanes 2 to 8), empty vectors (lane 2), human p53 (lanes 3 to 8), HPV-16 E6 (lanes 4 and 7), and/or human MDM2 (lanes 5 and 8). Eight hours after transfection, 5 ng of LMB per ml was added to half of the samples (lanes 6 to 8). At 24 h posttransfection, lysates were made and luciferase activity in each sample was determined in order to normalize for transfection efficiency. Equal relative light units were run on SDS-PAGE, and p53 was visualized by immunoblotting as described in the legend to Fig. 1. E6-mediation degradation of p53 does not require MDM2, but is partially blocked by LMB. Molecular weights (MW) are shown to the left (thousands).
A model for the degradation of p53 by MDM2. MDM2, p53, CRM1, and RanGTP are predicted to form a ternary complex in the nucleus, which is transported to the cytoplasm through the nuclear pore. p53 is then targeted to cytoplasmic proteasomes, where it is degraded. CRM1, Ran (now in the GDP-bound form), and possibly MDM2 return to the nucleus to repeat the process. The addition of LMB prevents the formation of this complex in the nucleus so that p53 is not shuttled to the cytoplasm for degradation. As predicted by the model, p53 levels in the nucleus rise after LMB treatment.
References
- Barak Y, Gottlieb E, Juven-Gershon T, Oren M. Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential. Genes Dev. 1994;8:1739–1749. - PubMed
- Bueso-Ramos C E, Yang Y, deLeon E, McCown P, Stass S A, Albitar M. The human MDM-2 oncogene is overexpressed in leukemias. Blood. 1993;82:2617–2623. - PubMed
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