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Nuclear degradation of p53 occurs during down-regulation of the p53 response after DNA damage¹

Department of Pathology, State University of New York at Stony Brook, Stony Brook, New York 11794, USA

²Correspondence: Department of Pathology, Basic Science Tower L9, State University of New York at Stony Brook, Stony Brook NY 11794-8691, USA. E-mail: umoll{at}notes.cc.sunysb.edu

SPECIFIC AIMS

A key determinant of p53 activity is its protein level. The principal regulator of p53 stability is HDM2, an E3 ubiquitin ligase specific for p53. HDM2 mediates p53 degradation via the ubiquitin-26S proteasome pathway. It is now thought that p53 degradation occurs exclusively on cytoplasmic proteasomes, and hence has an absolute requirement for nuclear export of p53, which occurs via the CRM1 pathway. However, proteasomes are abundant in both the cytosol and nucleus. We examined whether and under what physiological circumstances p53 degradation might occur directly in the nucleus.

PRINICPAL FINDINGS

1. Endogenous p53 can be degraded in the nucleus
MDA 231 human breast cancer cells exhibit visible nuclear p53 levels in 100% of cells due to a p53 R280K mutation, rendering this system convenient for studying endogenous p53 degradation at the single cell level. Ectopic HDM2 completely and rapidly degrades endogenous nuclear p53 in 92% of transfected MDA 231 cells. Thus, we reasoned that after initial optical clearance by ectopic HDM2 (when export was still ongoing), addition of leptomycin B (LMB) should lead to a reaccumulation of p53 in the nuclei of most transfected cells if p53 degradation were to take place solely in the cytoplasm. However, reaccumulated p53 continues to be completely degraded in > 60% of cells despite a block in nuclear export by LMB. More important, when nuclear export was shut down before the onset of HDM2 expression, so that only nuclear proteasomes were available for degradation, 42% of HDM2 expressing cells still showed a complete disappearance of nuclear p53. On the other hand, 48% of HDM2-expressing cells are resistant to p53 degradation, indicating their reliance on cytoplasmic degradation. Identical results with 45% degradation were seen with MDA 468 breast cancer cells (p53 R273H). In contrast, the RING finger mutant cannot degrade p53, irrespective of the absence or presence of LMB.

2. Nuclear export blockade by HTLV1 Rex exhibits nuclear and cytoplasmic degradation of p53
To confirm the LMB data with a nonpharmacological mode of CRM1 export blockade, we used an improved version of HTLV1-Rex called NLS-Rex, an efficient competitive nuclear export inhibitor. The NES sequences of p53 and HDM2 are similar to HTLV1-Rex, and NLS-Rex has been previously used to block HDM2 export. Flag-tagged NLS-Rex (Flag-Rex) efficiently blocks nuclear export of NES-containing control protein GFP-IRF3 (Fig. 1 A) and GFP-HDM2 in MDA 231 cells. The GFP-HDM2 fusion protein locates to the nucleus and is fully functional for degrading p53 (Fig. 1B , top; C). Using triple color immunofluorescence, cells coexpressing GFP-HDM2 and Flag-Rex show complete nuclear p53 degradation in almost 40% of cells, revealing a component of nuclear p53 degradation. Only Rex-expressing cells with strictly nuclear GFP-HDM2 were scored to ensure that Flag-Rex was blocking export completely in these cells. Flag-Rex was expressed prior to GFP-HDM2, excluding the possibility that p53 degradation might have been caused by a delayed Flag-Rex expression and nuclear leakage; on the other hand, 50% of coexpressing cells exhibited persistence of nuclear p53, indicating their dependence on cytoplasmic proteasomes (Fig. 1B , 1C ).

View larger version (39K): [in this window] [in a new window]   Figure 1. Nuclear export blockade by an optimized HTLV1 Rex exhibits nuclear and cytoplasmic degradation of p53. A) Flag-Rex blocks nuclear export of NES-containing marker protein GFP-IRF3 in MDA 231 cells. Rex is detected by anti-Flag and TRITC-conjugated secondary antibody. Cells at the bottom are untransfected. B) Cotransfection of MDA 231 cells with GFP-HDM2 and Rex. Upper row: GFP-HDM2 fusion protein locates to the nucleus and is fully functional for degrading p53. Bottom row: Cells coexpressing GFP-HDM2 and Flag-Rex show complete nuclear p53 degradation in almost 40% of cells (indicated by a star and blue-green color when merged). On the other hand, 50% of cells exhibit resistance to nuclear p53 degradation (indicated by a cross and pale yellow color when merged). C) Quantitation of panel B: 3 independent experiments, SDs are shown. The number of HDM2-transfected cells for each group is indicated.

3. Cells coexpressing export-defective p53 and HDM2 retain partial competence for p53 degradation
To independently confirm the LMB and CRM1 blocking data, we next performed mutational studies disabling the NES signals of p53 and HDM2 and asked whether nuclear degradation of p53 still occurs. A loss-of-function mutation in the carboxyl-terminal NES of p53 (p53 L348,350A) is sufficient to confine p53 to the nucleus. When p53-deficient H1299 or SaOs-2 cells were cotransfected with wtp53 and wt HDM2, degradation of p53 was strongly promoted. When the nucleus-confined p53 L348,350A mutant (p53 NES) was coexpressed with wt HDM2, degradation of p53 NES was still promoted, albeit to a lesser degree than that of freely mobile wt p53. Finally, p53 NES was cotransfected with a mutant HDM2 plasmid (HDM2 NES) encoding an E3 ligase-competent but equally export-disabled HDM2 to exclude any possible shuttling of p53 by HDM2. This HDM2 NES again promoted the degradation of p53 NES, and did so to the same degree as wild-type HDM2.

4. Nuclear degradation of p53 occurs during down-regulation of the p53 response
To determine the physiological significance of nuclear p53 degradation, we monitored the decline of p53 levels during the recovery phase after a p53 response under conditions that severely restrict or abrogate the export ability of p53. Since HDM2 NES promotes degradation of the nuclear-confined p53 NES, it provides a genetic system that focuses on nuclear degradation. H1299 cells were cotransfected with p53 NES and HDM2 NES. After 24 h, cells were subjected to DNA damage for 2 h by 5 µM Camptothecin. After washing and transfer to fresh medium, cells were allowed to recover for 0–18 h (Fig. 2 A). At 6 h after DNA damage, p53 levels peaked despite the presence of HDM2 NES. This confirms the notion that DNA damage-induced p53 is resistant to HDM2 degradation due to amino-terminal phosphorylation of p53 that disrupts HDM2 binding, thereby stabilizing p53. After a recovery of 12 h, p53 NES levels had fallen to or below the levels present before DNA damage. Alternatively, H1299 cells were cotransfected with wtp53 and wtHDM2 for 24 h, followed by 2 h of DNA damage as above. After washing and transfer to fresh medium, Leptomycin B was added at the peak of p53 stabilization to block all nuclear export during the post-stress recovery phase. p53 was again degraded from its peak at 6 h to lower levels at 12 h and reached very low levels at 24 h (Fig. 2B ).

View larger version (39K): [in this window] [in a new window]   Figure 2. Nuclear degradation of p53 occurs during down-regulation of the p53 response after DNA damage. A) Post-stress degradation of nucleus-restricted p53 NES mutant protein in H1299 cells. H1299 cells were transfected with p53 NES and with or without a nucleus-confined HDM2 plasmid (HDM2 NES). Twenty-four hours later, cells were transiently treated with 5 µM Camptothecin for 2 h (lanes 3–6), transferred to fresh medium, and the recovery phase was monitored. Total cell lysates, normalized by GFP, were immunoblotted for p53 and HDM2. B) Post-stress degradation of wt p53 protein in H1299 during nuclear export blockade. H1299 cells were transfected with wtp53 and with or without wtHDM2 plasmid. Twenty-four hours later, cells were treated with 5 µM Camptothecin for 2 h (lanes 3–6) and transferred to fresh medium. Lanes 4–6: LMB was added at the peak time of p53 stabilization at 6 h (see panel A). The recovery phase was monitored as in panel A. Total recovery is the time after Camptothecin was removed; the corresponding LMB exposure at each time point is 6 h less. Shorter ECL exposures were deliberately chosen to better visualize differences in protein levels.

CONCLUSIONS AND SIGNIFICANCE

This is the first study addressing whether nuclear proteasomes are a determinant for controlling p53 stability and, if so, in which physiological situation this might occur. Our data strongly argue that nuclear proteasomes, in conjunction with cytoplasmic proteasomes, are indeed an important physiological compartment in regulating p53 stability during post-stress recovery. Once a cell has successfully dealt with a DNA damage event, the p53 pathway needs to be turned off quickly. Sole reliance on cytoplasmic export to rid the cell of doomed p53 requires additional time and energy in the form of Ran-GTP via the CRM1 pathway. The added ability to degrade p53 locally in the nucleus provides several advantages to the cell since it bestows a tighter and faster control during the down-regulatory phase, after an active p53 response is no longer needed or is even deleterious to cell survival. Additional degradative capacity in the nucleus might also be important because p53 NES signals are weak. In a quantitative comparison of 10 different Rev-type NES signals that export via the CRM1 receptor (MAPKK, PKI-, c-Abl, Ran-BP1, IB, Rev, FMRP, TFIIIA, HDM2, p53), p53 NES was the weakest.

There is no biological reason per se why nuclear export should be required for p53 degradation. 1) Ubiquitination, the precondition for degradation, clearly occurs locally in the nucleus and probably occurs exclusively in the nucleus. 2) The proteasomal machinery is soluble and freely mobile and relocates to its substrates, rather than substrates having to find the proteasome. The proteolytic core 20S proteasome, binds to two 19S ATPase regulatory complexes (caps) at each end of its barrel structure to form the complete 26S proteasome. This 26S structure carries out the ubiquitin-dependent proteolysis with high processivity, resulting in short peptides of 3 to 20 residues. 3) 26S proteasomes are ubiquitously and evenly distributed throughout the cell in both nucleus and cytoplasm. In summary, we propose to amend the current static model that HDM2-mediated degradation of p53 occurs exclusively in the cytoplasm and that nuclear export is an absolute prerequisite. In nonlethal outcomes of cellular stress, when DNA damage has been repaired and the active p53 response needs to be down-regulated quickly to resume normal homeostasis, both nuclear and cytoplasmic proteasomes are being recruited to efficiently degrade the elevated p53 levels (Fig. 3 ).

View larger version (51K): [in this window] [in a new window]   Figure 3. Model for nuclear and cytoplasmic sites of HDM2-mediated p53 degradation. HDM2, an E3 ubiquitin ligase for p53, mediates p53 degradation via the ubiquitin–26S proteasome pathway. Proteasomes are abundant in both cytosol and nucleus. During the down-regulation of a p53 response after DNA damage, the cell takes advantage of both compartments to degrade p53. The ability of local nuclear destruction adds important control and flexibility when turning the p53 pathway off.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0617fje; to cite this article, use FASEB J. (January 14, 2002) 10.1096/fj.01-0617fje

This Article Full Text (PDF) All Versions of this Article: 16/3/420 01-0617fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by SHIRANGI, T. R. Articles by MOLL, U. M. Search for Related Content PubMed PubMed Citation Articles by SHIRANGI, T. R. Articles by MOLL, U. M.