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Department of Pathology, State University of New York at Stony Brook, Stony Brook, New York, USA
1Correspondence: Department of Pathology, State University of New York at Stony Brook, Stony Brook, NY 11794, USA. E-mail: umoll{at}notes.cc.sunysb.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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-IR, camptothecin, or cisplatinum. Moreover, significant nuclear degradation of endogenous p53 and HDM2 occurs in isolated nuclear fractions prepared from these recovering cells. Furthermore, nuclear proteasomes efficiently degrade ubiquitinated p53 in vitro. Our data indicate that in nonlethal outcomes of cellular stress, when DNA damage has been successfully repaired and the active p53 response needs to be down-regulated quickly to resume normal homeostasis, both nuclear and cytoplasmic proteasomes are recruited to efficiently degrade the elevated p53 and HDM2 protein levels. The physiological significance of local nuclear destruction lies in the fact that it adds tighter control and speed to switching the p53 pathway off.—Joseph, T. W., Zaika, A., Moll, U. M. Nuclear and cytoplasmic degradation of endogenous p53 and HDM2 occurs during down-regulation of the p53 response after multiple types of DNA damage.
Key Words: p53/HDM2 • ubiquitination • proteasomes • degradation • cytoplasm • nucleus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
4
5)
. The E3 ligase activity of HDM2 maps to a RING finger motif at the carboxyl terminus, which ubiquitinates p53 on multiple lysine residues throughout its carboxyl terminus (5)
. HDM2 is responsible for low p53 levels in unstressed cells and also for switching off a p53 stress response after cell damage is successfully repaired, due to an autoregulatory feedback loop in which activated p53 stimulates HDM2 transcription.
A long held notion stated that the cytoplasm is the exclusive site of p53 degradation, and therefore nuclear export of p53 was thought to be a prerequisite for its delivery to cytoplasmic proteasomes (6
7
8)
. This model is based on two pieces of evidence. First, leptomycin B (LMB), a specific inhibitor of the CRM1 nuclear export receptor, induces nuclear accumulation of p53 (8
9
10)
. Second, the existence of two nuclear export signals (NES) within p53, which was interpreted to indicate obligatory export for the sole purpose of degradation (9
, 11)
. The carboxyl-terminal NES, located within the tetramerization domain, is masked when p53 forms a transcriptionally active tetramer but unmasked when p53 dissociates into a monomer or dimer (9)
. The amino-terminal NES is blocked by DNA damage-induced phosphorylation, suggesting it operates only in unstressed cells (11)
. The MDM2 RING finger domain, but not the NES signal of MDM2, is required for efficient export of p53 to the cytoplasm (12
, 13)
. The likely mechanism is that MDM2 monoubiquitinates all available lysine residues in the carboxyl terminus of p53 (14
, 15)
, thereby revealing the NES in the adjacent tetramerization domain and allowing interaction with the CRM1 export machinery (15)
. However, one important caveat of this "obligatory export model" is that it is based largely on studies using ectopic p53 proteins, often as GFP-p53 fusion proteins, expressed at excessively high levels. The second caveat is that the interpretation of the LMB results is unclear, since LMB is not only a powerful export blocker of CRM1 with respect to Rev-like NES proteins such as p53, but is most likely also a specific stress signal for p53, which in itself would induce marked p53 accumulation in the nucleus as well as p53 transcriptional activation (10
, 16)
.
Importantly, 26S proteasomes are equally abundant in both cytosol and nucleus (17
18
19
20)
. Moreover, ubiquitination of p53, the precondition for its degradation, clearly occurs locally in the nucleus (21)
; in fact, the nucleus is probably the exclusive site for this modification (12
, 13)
. Taken together, there is therefore no a priori biological necessity why nuclear export should be required for p53 degradation. This prompted us to address the possibility that p53 degradation might occur directly in the nucleus. In a recent preliminary study, we monitored p53 degradation of tumor cells with constitutively stable mutant p53 after ectopic overexpression of HDM2 at single cell resolution and in the presence of the CRM1 inhibitors LMB and HTLV1-Rex protein (22)
. We found that the nucleus constitutes a significant proteasomal compartment for HDM2-mediated degradation and operates in parallel to the cytoplasmic compartment. Moreover, p53 null cells cotransfected with export-defective NES mutants of both p53 and HDM2 retain partial competence for p53 degradation. Importantly, when those null cells were subjected to DNA damage, nuclear degradation of ectopic p53 occurred during the poststress recovery phase (22)
. Based on these data, we hypothesized that local nuclear destruction could add important control in turning off the p53 pathway after high p53 levels are no longer needed or even deleterious to cell survival. We tested whether this occurs physiologically in nontransfected cells harboring endogenous wild-type p53 and HDM2 and determined the significance of this regulation. Our results indicate that nuclear degradation of p53 and HDM2 does occur in cells during down-regulation of the p53 response after multiple types of DNA damage.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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by PCR as described previously (22)
.
DNA damage treatment
Cells were plated in 60 mm plates and subjected to the following DNA damage treatments. camptothecin (5 µM) or cisplatinum (25 µM) were added to the culture medium for 2 h. Alternatively, cells were exposed to 1500 J/m2 of UVB (Stratagene UV Cross-linker) or 10 Gy of -IR (Cs137 source). Cells were then washed and placed into fresh medium and incubated for another 4 h, during which the p53 response occurred (here called stabilization). Since maximum stabilization of p53 and HDM2 occurred after 4 h for all cell and stress types (Fig. 1
A), the subsequent decay of p53 and HDM2 was monitored from that point on in all experiments. To impose nuclear export blockade of these maximally stabilized protein pools, leptomycin B (20 nM) was added into the medium at that time. Alternatively, cells were transfected with NLS-Rex prior to DNA damage. For immunofluorescence, cells were seeded into 8-well chamber slides and grown overnight to 80% confluence. NLS-Rex construct or empty vector (0.6 µg each) was transfected using 2.6 µL of FuGene 6 Transfection Reagent (Roche, Nutley, NJ, USA) per well. For Western analyses, cells were plated in 60 mm dishes and grown to 80% confluence before transfection. To monitor only the fate of previously stabilized pools of p53 and HDM2 proteins, cycloheximide (40 µg/mL) was added to prevent new synthesis. Cells were harvested after 1, 2, 4, and 16 h and lysed to generate whole cell lysates. Alternatively, cells were first fractionated into nuclear and cytoplasmic compartments according to the manufacturer’s instructions (Pierce), followed by lysis.
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Antibodies and immunoblot analysis
Equal amounts of protein were subjected to 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Immunoblots were visualized by Supersignal Chemiluminescent System (Pierce). Endogenous p53 was detected with monoclonal DO-1 and endogenous HDM2 was detected with monoclonal IF-2 (both from Oncogene Science, Cambridge, MA, USA). Endogenous Lamin A was used as a nuclear contamination marker and vimentin as a cytosolic contamination marker (both from Chemicon, El Segundo, CA, USA). NLS-Rex expression was detected by monoclonal M2 against its Flag-tag (Sigma) and respective immunoblot loading was adjusted for equal Flag intensity.
In vitro p53 ubiquitination assay
A pGEX-based GST-MDM2 expression plasmid (gift of Dr. A. Weissman, NCI) was expressed in BL21 cells. Recombinant protein was purified over a glutathione-Sepharose column (Pharmacia). Untagged mouse baculoviral wtp53 was purified over a MonoQ column and appeared as a single 53 kDa band on silver gel (not shown). To generate ubiquitinated p53 as substrate, 100 ng of purified p53 was mixed with recombinant E1 (200 ng), E2 (UbcH5B) (150 ng), ubiquitin (10 µg) (all from Oncogene), and 500 ng of purified GST-MDM2 for 4 h in a total volume of 30 µL ubiquitination buffer at 30°C in an Eppendorf Thermomixer at 800 rpm as described by Fang et al. (24)
. The reaction was stopped by adding sample buffer and analyzed by immunoblot with DO-1 antibody. Under these conditions, ubiquitinated p53 appears as a group of tight bands of 
90 kDa (25)
. Unstressed RKO cells were either lysed as whole cells (‘crude’) or carefully separated into nuclear and cytoplasmic fractions as above, then lysed. Fractions lacked detectable cross contamination as judged by Lamin A and vimentin. Two hundred micrograms total protein of either crude lysate or of each fraction (source of proteasomes) were mixed with 1 ng of ubiquitinated p53 for 3 h at 30°C in 50 mM Tris pH 7.4, 2 mM ATP, 5 mM MgCl2, 2 mM DTT in a total volume of 200 µL. Duplicate vials also contained the proteasome inhibitor Mg132 (30 µM) to block in vitro degradation. Samples were analyzed by DO-1 immunoblots.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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). As expected, at the 4 h time point the majority of detectable p53 locates to the nucleus, indicated by a bright nucleus by immunofluorescence and only faint staining of the cytoplasm (data not shown). To monitor possible degradation of endogenous p53 and HDM2 in the nucleus, leptomycin B was added at maximum stabilization (4 h) to block nuclear export. One hour later, cycloheximide was added, which prevents neosynthesis and ensures that we monitor only the fate of previously stabilized pools of p53 and HDM2 proteins. The decline of steady-state levels of endogenous p53 and HDM2 were subsequently analyzed after 1, 2, 4, and 16 h in whole cell lysates of U2OS and RKO cells (Fig. 1C, E
). The results clearly show that endogenous p53 and HDM2 undergo degradation in the nucleus in the presence of a nuclear export blockade. In RKO cells, 71% of p53 is degraded after 1 h compared with its peak level (set as 100%), almost reaching prestress levels (Fig. 1E
, compare lanes 1 and 3) and is undetectable at 16 h. In U2OS cells, 52% of p53 is degraded after 4 h compared with its peak level and is undetectable at 16 h (Fig. 1C
, left panel). Interestingly, in both cell lines, HDM2 degradation in the nucleus is faster than that of p53, with 100% degradation after 1 h in RKO cells (Fig. 1E
) and 100% degradation after 2 h in U2OS cells (Fig. 1C
, left panel). To further demonstrate that LMB was reliably working as a CRM1 nuclear export inhibitor throughout the experiment, LMB-containing medium was collected from those cells after 16 h and reapplied to untreated RKO and U2OS cells for an additional 8 h. As shown in Fig. 1C, E
, middle and right panels, this medium stabilized p53 and HDM2 in both cell types compared with untreated control cells, demonstrating that LMB was still active at this late time point. Moreover, when we compared poststress p53 degradation in the presence or absence of LMB, the kinetic profiles were very similar (Fig. 1C
, left and right panels; compare both 4 h time points), supporting the notion that significant p53 degradation occurs in the nucleus and does not depend on transport to the cytoplasm. We next used an alternative mode of CRM1 export blockade. HTLV1-Rex protein contains a strong NES that is homologous to the prototype NES in the HIV-1 Rev protein and efficiently exports viral mRNAs out of the nucleus. An improved Rex protein, deleted in its RNA binding domain but retaining its NES and fused to the nuclear localization signal from SV40 large T-antigen, is a strong CRM1 export inhibitor (23)
. This "NLS-Rex" rapidly shuttles in and out of the nucleus. Overexpressing this innocuous inhibitor efficiently prevents other NES-containing proteins from binding to CRM1 receptors, thereby inhibiting their export (23)
. The NES sequences of p53 and HDM2 are very similar to Rex (9)
and NLS-Rex has been used before to block endogenous p53 and HDM2 export (6
, 22)
. Importantly, when p53 export was inhibited by NLS-Rex, nuclear p53 degradation took place and did so with very similar kinetics, as already seen with the LMB export blockade (Fig. 1D
). To further confirm that LMB and NLS-Rex are both blocking nuclear protein export under the experimental conditions used in this study, we used an unrelated protein, interferon regulatory factor 3 (IRF-3), which shuttles in and out of the nucleus via CRM1 and is known to be retained in the nucleus after LMB treatment (26)
. As shown in Fig. 1B
, endogenous IRF3, which is primarily cytoplasmic in untreated U2OS cells (upper left panel), becomes largely nuclear with LMB treatment (middle left panel). Likewise, IRF3 becomes largely nuclear with coexpressed NLS-Rex (bottom left panel). Also, as expected, endogenous p53 becomes locked into the nucleus by LMB and NLS-Rex (middle and bottom right panels). Thus, taken together, enforced nuclear export blockade reveals that the nuclear degradative capacity alone is sufficient to revert maximally stabilized p53 and HDM2 levels back to prestress levels within 16 h or less.
To more rigorously examine the nuclear compartment with respect to degrading endogenous p53 and HDM2 proteins during poststress recovery, we applied the same treatment and recovery scheme as in Fig. 1
, but at each time point we subjected cells to nuclear/cytoplasmic fractionations before analysis. As an extra precaution, LMB was added again at maximum stabilization (4 h) to prevent any potential nuclear export during the process of fractionation, although the possibility of this occurring was rather remote (Fig. 2
A, C–E). Alternatively, nuclear export blockade was achieved by prior transfection with NLS-Rex (Fig. 2B
). The purity of the fractions was excellent since neither the nuclear marker Lamin A nor the cytoplasmic marker vimentin showed any cross contamination (Fig. 2A-E
and Fig. 3
A–C). As shown in Fig. 2A, B
, the degradation profiles in the nuclear fractions of camptothecin-treated U2OS cells mirrored that of whole cell lysates of U2OS (see Fig. 1C
). Four hours after maximum stabilization, 72% of p53 and 100% of HDM2 were degraded in the nuclear fraction (Fig. 2A
). Similar results were obtained in the nuclear fractions of camptothecin-treated RKO cells, where 74% of p53 was degraded after 4 h (Fig. 3A
).
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Moreover, p53 and HDM2 induced by cisplatinum, UV, and -IR of U2OS and RKO cells were significantly degraded in the nuclear fractions of these cells (Fig. 2C-E
, Fig. 3B, C
and data not shown). For example, 4 h after UVB treatment of U2OS cells (1500 J/m2), maximally stabilized nuclear p53 levels had almost completely returned to normal levels (Fig. 2D
, compare lane 5 with prestress level in lane 1). Nuclear degradation in U2OS cells after camptothecin, cisplatinum, and -IR took longer than 4 h but was completed at the latest 16 h after maximum stabilization in all cases (Fig. 2A-C, E
). Cell type variations exist, since nuclear degradation of p53 in RKO cells was somewhat slower than that of p53 in U2OS cells, as seen in camptothecin treatment (compare Fig. 3A
with Fig. 2A
), UVB treatment (compare Fig. 3B
with Fig. 2D
), and -IR (compare Fig. 3C
with Fig. 2E
). Strikingly, in all cases nuclear HDM2 degradation was faster than nuclear p53 degradation (Fig. 2A
and panels C–E, Fig. 3A-C
). Moreover, the completeness of nuclear HDM2 degradation was indicated by the fact that nuclear HDM2 levels fell below those of unstressed cells (Fig. 2A, C-E
and Fig. 3A-C
). This effect was likely promoted by the inhibition of new HDM2 synthesis by cycloheximide. Of note, regardless of cell type, treatment type, or time point analyzed, we failed to detect any p53 and HDM2 protein in the cytoplasmic fractions, even at the peak levels. This is expected for time points after DNA damage occurred, since p53 localization is known to be entirely nuclear after cellular stress. It is surprising, however, that even the cytoplasmic fractions of untreated samples failed to show any detectable p53 and HDM2, suggesting that the steady-state levels of p53 and HDM2 in the cytoplasm of unstressed cells are very low.
Nuclear proteasomes efficiently degrade recombinant ubiquitinated p53 in vitro
To specifically demonstrate that nuclear proteasomes are capable of degrading p53 and that the degradative efficiency of nuclear and cytoplasmic proteasomes is roughly comparable, we performed an in vitro degradation assay using recombinant ubiquitinated p53 as substrate and either nuclear or cytoplasmic fractions as proteasome sources. First, ubiquitinated p53 was generated in vitro by incubating baculoviral mouse wild-type p53 with recombinant E1, E2 (UbcH5B), GST-MDM2, and ubiquitin moieties for 4 h. Because MDM2 was in excess, Ub-p53 appeared as a group of tight bands of 90 kDa, corresponding to complete ubiquitination of all lysine residues at the carboxyl terminus of p53 that are targeted by MDM2 (Fig. 4
A, upper bands in right lane). As shown in Fig. 4B
, 200 µg of whole cell lysates of RKO cells (‘crude’), containing both cytoplasmic and nuclear proteasomes, markedly degraded Ub-p53 (compare lanes 3 and 4). Degradation was completely blocked by proteasome inhibitor MG 132, demonstrating proteasome specificity (compare lanes 3 and 5). Likewise, cytoplasm-derived proteasomes contained in 200 µg cytoplasmic fractions of RKO cells were also able to degrade Ub-p53, and degradation was inhibited by MG 132 (compare lanes 3, 6, and 7). Importantly, the nucleus-derived proteasomes contained in 200 µg nuclear fractions of RKO cells degraded Ub-p53 equally well. Again, proteasomal specificity was shown by abrogation with MG 132 (compare lanes 3, 8, and 9). The same amount of Ub-p53 (1 ng) was used in all reactions. These two fractions were related to the relative cell numbers used to generate them, i.e., 0.5 x 106 cells for the nuclear and 0.38 x 106 cells for the cytoplasmic fraction, indicating that the nuclear p53 degradation capacity of RKO cells was 25% lower than their cytoplasmic capacity. Nevertheless, the overall capacity is similar.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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ionizing radiation. Future work will clarify whether nuclear degradation of p53 occurs under additional physiologic conditions such as in unstressed cells or during recovery from hypoxia or ribonucleoside depletion.
26S proteasomes are ubiquitously and evenly distributed throughout the cell in the nucleus and cytoplasm (17
18
19
20)
. Within each of these two compartments, proteasomes diffuse rapidly, while between compartments fully assembled proteasomes are transported slowly and unidirectionally from the cytoplasm into the nucleus but not in the reverse direction (17)
. Moreover, the proteasomal machinery is freely mobile and relocates to its substrates (17
, 27)
. This general cell biology of proteasomes supports our conclusion that both compartments contribute to rapid degradation, rather than exclusively the cytoplasmic compartment, as previously thought. Work by two other groups using transfection-based approaches also support our findings. Lohrum et al. found that although p53 nuclear export was enhanced by overexpression of CRM1, it did not result in the expected enhanced p53 degradation. In fact, overexpression of CRM1 reduced the sensitivity of p53 to MDM2-mediated degradation (15)
. Moreover, Xirodimas et al. found that as long as cotransfected MDM2 and p53 are co-compartmentalized, be it in the nucleus or the cytoplasm, MDM2 promotes ubiquitination and degradation of p53. Thus, efficient degradation of ectopic p53 occurs in the absence of either nuclear import or export (28)
. Taken together, these data support the notion that nuclear export and degradation of p53 are not correlated and may be regulated separately.
Our results show that during the down-regulation of a p53 response after DNA damage, the cell takes advantage of both compartments to degrade p53 and HDM2. After 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 is relatively slow and consumes additional energy in the form of Ran-GTP for the CRM1 pathway. The added ability to degrade p53 locally in the nucleus provides several advantages to the cell since it bestows a tighter regulatory control during the turn-off phase, after an active p53 response is no longer needed or even deleterious to cell survival. Additional degradative capacity in the nucleus might also be important because p53 NES signals are weak (29)
. In a quantitative comparison of 10 different Rev-type NES signals that export via the CRM1 receptor (MAPKK, PKI-
, c-Abl, Ran-BP1, I
BRev, FMRP, TFIIIA, HDM2, and p53, listed by decreasing rank), their relative export rates in vivo varied considerably (29)
. The carboxyl-terminal NES of human p53 was the weakest and associated with the slowest export rate in a live GFP-NES export assay compared with MAPKK and PKI-, which were ninefold stronger. Moreover, the p53-NES sequence generated detectable cytoplasmic fluorescence only when import was simultaneously blocked and in just one of two cell lines tested. In contrast, the other nine NES sequences worked in both cell lines without requiring an import block (28)
. These arguments together lend rational support to the notion that both compartments mediate p53 degradation.
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ACKNOWLEDGMENTS |
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Received for publication September 25, 2002. Accepted for publication May 8, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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