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Cancer Research 67, 1959-1969, March 1, 2007. doi: 10.1158/0008-5472.CAN-06-3602
© 2007 American Association for Cancer Research
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Molecular Biology, Pathobiology, and Genetics

The p53 Isoform {Delta}p53 Lacks Intrinsic Transcriptional Activity and Reveals the Critical Role of Nuclear Import in Dominant-Negative Activity

Wan Mui Chan and Randy Y.C. Poon

Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong

Requests for reprints: Randy Y.C. Poon, Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong. Phone: 852-2358-8703; Fax: 852-2358-1552; E-mail: bcrandy{at}ust.hk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The transcription factor p53 is one of the most frequently mutated tumor suppressors. Recent progress has unraveled several novel isoforms of p53. Intriguingly, one of the p53 isoform, {Delta}p53, which lacks part of the DNA binding domain, was reported to be transcriptionally active toward some p53 target genes and is critical for the intra–S phase checkpoint. Here, we show that, in contrast to full-length p53, ectopically expressed {Delta}p53 neither transactivated the promoters of p21CIP1/WAF1 or murine double minute-2 (MDM2) nor repressed the cyclin B1 promoter in unstressed H1299 cells. Due to the deletion of a nuclear localization signal, {Delta}p53 was not imported into the nucleus. Engineering of nuclear localization signals to {Delta}p53 restored nuclear accumulation. However, the nuclear-targeting {Delta}p53 remained inactive, indicating that the lack of intrinsic activity of {Delta}p53 was not simply due to subcellular localization but to its incomplete DNA binding domain. Similar to p53, {Delta}p53 was subjected to MDM2-mediated ubiquitination/proteolysis. The cytoplasmic localization of {Delta}p53 correlated with the instability of the protein because forcing {Delta}p53 into the nucleus increased its stability. Although {Delta}p53 could form a complex with p53 and stimulated the cytoplasmic retention of p53, it was not a robust inhibitor of p53. Targeting {Delta}p53 into the nucleus enhanced the dominant-negative activity of {Delta}p53. These observations underscore the critical role of subcellular localization in the dominant-negative action of p53. [Cancer Res 2007;67(5):1959–69]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Loss of the p53 tumor suppressor function is one of the most common steps in tumorigenesis. Germ line mutations of p53 (TP53) are present in cancer-prone families with Li-Fraumeni syndrome (1), and somatic mutations are found in more than half of all cancer cases (2).

The p53 gene encodes a protein with a central DNA binding domain, flanked by an NH2-terminal transactivation domain and a COOH-terminal tetramerization domain (3). The active form of p53 is a tetramer of four identical subunits, consisting of a dimer of a dimer (4). Consistent with its tetrameric state, p53 binds DNA sites that contain four repeats of the pentamer sequence motif 5'-Pu-Pu-Pu-C-A/T-3' (Pu is purine). The majority of the mutations in p53 are missense point mutations clustered in the DNA binding domain (5). The structure of the DNA binding domain consists of a large ß-sandwich that acts as a scaffold for three loop-based elements that contact the DNA (6). Importantly, the residues most frequently mutated in cancers are all at or near the protein-DNA interface, and over two thirds of the missense mutations are within the DNA binding loops (7).

Many studies have detailed the role of p53 as a transcription factor. A myriad of genes are transactivated by p53 and many of which are believed to be underlie the antiproliferative functions of p53 (8), including genes whose products are critical for cell cycle arrest (p21CIP1/WAF1, 14-3-3{sigma}, and GADD45) and apoptosis (BAX, NOXA, and PUMA). Given the critical role of p53 in controlling cell proliferation, it is not surprising that its levels and activities are tightly regulated. Under normal conditions, murine double minute-2 (MDM2; also one of the transcriptional targets of p53) binds to the transactivation domain of p53 and abrogates p53-mediated transcription. MDM2 also shuttles p53 out of the nucleus and targets p53 for ubiquitin-mediated proteolysis, keeping p53 at a low level under unstressed conditions (9). Other ubiquitin ligases including MDMX (10), PIRH2 (11), and COP1 (12) also seem to contribute to p53 ubiquitination. On DNA damage or other stresses, p53 is phosphorylated by ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) at Ser15 (13) and CHK1/CHK2 at Ser20 (14, 15). Phosphorylation of these residues (as well as other NH2-terminal residues by various kinases) disrupts the p53-MDM2 interaction and promotes p53 accumulation. Besides ubiquitination and phosphorylation, p53 is also regulated by other posttranslational modifications, including acetylation by CREB binding protein/p300 at multiple COOH-terminal lysine residues, neddylation, and sumoylation (16).

Two p53-related genes, p63 (TP63) and p73 (TP73), encode proteins that share high sequence homology with p53, particularly at the DNA binding domain. This enables p63 and p73 to also transactivate p53-responsive genes, causing cell cycle arrest and apoptosis (17). A notable feature of p63 and p73 is that both genes express a large number of isoforms (17). Human p63 encodes at least six different isoforms: three are derived from alternative splicing of the COOH terminus (TAp63{alpha}, TAp63ß, and TAp63{gamma}) and three are transcribed from an alternative promoter located in the intron 3, producing proteins lacking the NH2-terminal transactivation domain ({Delta}Np63{alpha}, {Delta}Np63ß, and {Delta}Np63{gamma}; ref. 18). Human p73 expresses at least seven alternatively spliced COOH-terminal isoforms ({alpha}, ß, {gamma}, {delta}, {varepsilon}, {zeta}, and {eta}) and at least four alternatively spliced NH2-terminal isoforms initiated at different ATG. Like p63, p73 can be transcribed from an alternative promoter located in the intron 3 ({Delta}Np73). Both {Delta}Np63 and {Delta}Np73 can bind DNA through p53-responsive element and can exert dominant-negative effects over p53, p63, and p73 activities either by competing for DNA binding sites or by direct protein-protein interaction (19).

Until recently, the prevailing view was that the structure of p53 gene is much simpler than that of p63 and p73. Recent progress, however, has unraveled that p53 also encodes several isoforms. These include p53ß (also called p53i9) and p53{gamma}, which are produced from alternative splicing from intron 9 and lack the COOH-terminal tetramerization domain (20, 21). In addition, NH2-terminally truncated isoforms ({Delta}40p53, {Delta}40p53ß, and {Delta}40p53 {gamma}) are derived from alternative splicing of intron 2 or by alternative initiation of translation (22, 23). Another type of NH2-terminally deleted isoforms ({Delta}133p53, {Delta}133p53ß, and {Delta}133p53{gamma}) is transcribed from an internal promoter located in intron 4 (24). Similar to {Delta}Np63 and {Delta}Np73, both {Delta}40p53 (23) and {Delta}133p53 (24) have dominant-negative effect on wild-type p53 transcriptional activity and p53-mediated apoptosis. Furthermore, {Delta}40p53 can modify the subcellular localization of p53 and prevent p53 degradation by MDM2 (23).

Recently, Rohaly et al. (25) discovered that a novel p53 isoform, denoted as {Delta}p53, is generated by alternative splicing between exon 7 and exon 9. Sixty-six residues in the DNA binding domain of p53 are absent in {Delta}p53 ({Delta}257–322). Paradoxically, {Delta}p53 was reported to be transcriptionally active toward CIP1/WAF1 and 14-3-3{sigma}, but not MDM2, BAX, and PIG3. It was also reported that {Delta}p53 is expressed in several cell lines and is an essential element of the ATR-mediated intra–S phase checkpoint.

The presence of activity from {Delta}p53 is somewhat intriguing as the isoform contains an incomplete DNA binding domain (see Fig. 1A ). As {Delta}p53 still retains the tetramerization domain, it is more likely that {Delta}p53 can form tetramers with wild-type p53 and acts in a dominant-negative manner. Extensive data from studies in cell culture suggest that many missense mutant p53 can inhibit the transactivation of target genes. Mutated p53 present within a tetramer is thought to abolish the DNA binding capacity of the entire complex. This has the important implication that a heterozygous mutation in p53 could result in the functional inactivation of cellular p53. We have previously shown that DNA binding–defective p53 mutants can impair the transcriptional activity of p53, albeit rather ineffectively: at least three mutants are required to inactivate a tetramer (26). In marked contrast, NH2-terminally truncated p53 is a very potent inhibitor of p53: one N{Delta} subunit per tetramer is sufficient to abolish the transcriptional activity.


Figure 1
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  		Figure 1. {Delta}p53 interacts with p53 but transactivates p53-responsive promoters ineffectively. A, schematic diagram of the p53 constructs used in this study. The positions of the various functional elements of human p53 are shown to scale. All constructs, except {Delta}p53-NLS, are FLAG tagged at the NH2 terminus. Also used in this study were untagged p53 and {Delta}p53-NLS, HA-tagged p53, and V5-tagged p53 and {Delta}p53. B, {Delta}p53 does not activate p21CIP1/WAF1 and MDM2. H1299 cells were transfected with control vector or plasmids expressing p53, {Delta}p53, R249S, or R273H mutants as indicated. Cell extracts were prepared at 24 h after transfection and the abundance of p21CIP1/WAF1 and MDM2 was detected by immunoblotting (left). The recombinant p53 and {Delta}p53 were detected by immunoblotting for the FLAG tag. Equal loading of lysates was confirmed by immunoblotting for tubulin. Cells were also cotransfected with plasmids expressing luciferase reporters under the control of p21CIP1/WAF1 promoter or MDM2 promoter and a Renilla luciferase-expressing construct. The luciferase activity was measured, normalized with the Renilla luciferase activity to correct for variations in transfection efficiency between samples, and plotted as a percentage of p53 (right). Columns, mean of three independent experiments; bars, SD. C, {Delta}p53 can bind to wild-type p53. HA-tagged p53 was coexpressed with FLAG-{Delta}p53 in H1299 cells. Cell extracts were prepared and 100 µg were subjected to immunoprecipitation with either control normal rabbit serum (NRS) or FLAG antiserum. The immunoprecipitates were immunoblotted for HA. The blot was then probed for FLAG to verify the immunoprecipitation. Total cell lysates (10 µg) were applied to indicate the input. D, expression of p53 targets is not attenuated by {Delta}p53. Constant amount of FLAG-p53 and increasing amount of FLAG-{Delta}p53 were expressed in H1299 cells as indicated. At 24 h after transfection, cell extracts were prepared and the abundance of p21CIP1/WAF1 and MDM2 was detected by immunoblotting. The expression of FLAG-tagged p53 and {Delta}p53 was confirmed by immunoblotting for FLAG. Tubulin analysis was included to assess protein loading and transfer.

 
In this study, we explored whether {Delta}p53, like the other DNA binding mutants of p53, has the potential to interact with p53 and act in a dominant-negative manner. We found that, in contrast to full-length p53, {Delta}p53 did not transactivate the promoters of p21CIP1/WAF1 or MDM2. Furthermore, {Delta}p53 was ineffective in impairing the activity of p53. Significantly, our data revealed that {Delta}p53 was not imported into the nucleus. The cytoplasmic localization of {Delta}p53 correlated with the short half-life of the protein through ubiquitin-mediated proteolysis. Finally, whereas forcing {Delta}p53 into the nucleus did not activate the transcriptional activity of {Delta}p53 per se, this enhanced the dominant-negative activity of {Delta}p53. These observations underscore the critical role of nuclear localization in the dominant-negative action of p53.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials. All reagents were obtained from Sigma-Aldrich (St. Louis, MO) unless stated otherwise.

DNA constructs. Human p53 in pRcCMV (27), MDM2 in pCMV (28), pLINX (29), luciferase reporters under the control of p21CIP1/WAF1 promoter (30) or MDM2 promoter (27), and hemagglutinin (HA)-ubiquitin in pUHD-P2 (31) were obtained from sources as previously described. Constructs for HA-p53, FLAG-p53, p53 (R249S), and p53 (R273H) were as previously described (26). Cyclin B1 promoter-luciferase reporter was a generous gift from Dr. Denise Galloway (Fred Hutchinson Cancer Center, Seattle, WA). FLAG-p53 in pUHD-P1 (26) was amplified by PCR with a vector forward primer and 5'-CTTCTAGAGTGATGATGGTGAGGATGGGCCT-3'; the PCR product was cut with NheI-XbaI and ligated into pUHD-P1 (32) to generate FLAG-p53(C{Delta}257) in pUHD-P1. FLAG-p53 in pUHD-P1 was amplified by PCR with 5'-CCTCTAGATGGAGAATATTTCACCC-3' and a vector reverse primer; the PCR product was cut with XbaI-BamHI and ligated into FLAG-p53(C{Delta}257) in pUHD-P1 to create FLAG-{Delta}p53. This construct was then amplified by PCR with a vector forward primer and 5'-TTTCTCGAGTAAGTCTGAGTCAGGCCCTT-3' (p53-XhoI reverse primer); the PCR product was cut with NcoI-XhoI and ligated into pCMV/myc/nuc (Invitrogen, Carlsbad, CA) to create {Delta}p53(N{Delta}159)-nuclear localization signal (NLS)-myc. The NcoI-BamHI (the BamHI site was introduced with a primer at the myc tag) fragment was then put into pUHD-P1 to generate FLAG-{Delta}p53(N{Delta}159)-NLS-myc in pUHD-P1. The NcoI-NcoI fragment from p53 cDNA was ligated into NcoI-cut {Delta}p53(N{Delta}159)-NLS-myc in pCMV/myc/nuc to create {Delta}p53-NLS-myc in pCMV/myc/nuc. This construct and full-length p53 were amplified by PCR with 5'-CGAATTCCATGGAGGAGCCGCAGT-3' (p53-EcoRI forward primer) and p53-XhoI reverse primer; the PCR products were cut with EcoRI-XhoI and ligated into pcDNA6/V5-HisA (Invitrogen) to create {Delta}p53-V5-His and p53-V5-His in pcDNA6/V5-HisA, respectively. FLAG-{Delta}p53(N{Delta}159) was obtained by removing the NcoI-NcoI fragment from FLAG-{Delta}p53 in pUHD-P1.

Cell culture. H1299 cells (non–small-cell lung carcinoma; ref. 33) were obtained from the American Type Culture Collection (Rockville, MD). Cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum (Invitrogen) in a humidified incubator at 37°C in 5% CO2. Cycloheximide (10 µg/mL), doxycycline (1 µg/mL), and G418 (1 mg/mL) were used at the indicated concentrations. UV radiation was delivered with UVB (290–320 nm) erythemal tubes (Philips, Eindhoven, the Netherlands). The UV dose was calibrated with a UV meter from InternationalLight (Peabody, MA). The medium and the lid of the plate were removed before the cells were irradiated. Transfection was carried out with a calcium phosphate precipitation method (34). The amount of total DNA transfected was adjusted to the same level with blank vectors. H1299 cells were transfected with pLINX (a plasmid expressing the tTA transactivator; ref. 35) and grown in medium containing G418. After ~2 weeks of selection, single colonies were isolated and tested for inducible gene expression using doxycycline. Cell-free extracts were prepared as previously described (36). The protein concentration of cell lysates was measured with the bicinchoninic acid protein assay system (Pierce, Rockford, IL) using bovine serum albumin as a standard.

Transactivation assays. The transcriptional activity of p53 was assayed by transfecting cells with a promoter-luciferase (firefly) reporter construct and a Renilla reniformis luciferase construct. The activities of the two luciferases were analyzed with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). The activity of the firefly luciferase was normalized with that of the Renilla luciferase.

Ubiquitination assays. In vivo ubiquitination assays were done as previously described (31). Briefly, constructs expressing FLAG-tagged proteins were cotransfected with HA-ubiquitin in pUHD-P2. The cells were treated with 50 µmol/L of LLnL for 6 h before harvested. Cell extracts prepared from the transfected cells were immunoprecipitated with either normal rabbit serum or FLAG antiserum. The presence of HA-ubiquitin–conjugated proteins in the immunoprecipitates was detected by immunoblotting with the anti-HA monoclonal antibody 12CA5.

Fractionation. After harvest and washing with PBS, the cells were resuspended in 600 µL of buffer [10 mmol/L HEPES (pH 7.4), 1 mmol/L EDTA, and 1 mmol/L DTT] supplemented with protease inhibitors (2 µg/mL aprotinin, 15 µg/mL benzamidine, 1 µg/mL, leupeptin, 10 µg/mL pepstatin, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL soybean trypsin inhibitor) and incubated at 4°C for 10 min. The cells were then homogenized with 10 strokes in a tight pestle Wheaton Dounce homogenizer (Millville, NJ). The lysates were centrifuged at 240 x g for 5 min and the supernatant was collected (cytoplasmic fraction). The pellets were then washed thrice with buffer [10 mmol/L HEPES (pH 8), 50 mmol/L NaCl, 25% glycerol, and 0.1 mmol/L EDTA], centrifuged for 5 min, and resuspended in 30 µL of buffer [10 mmol/L HEPES (pH 8), 350 mmol/L NaCl, 25% glycerol, and 0.1 mmol/L EDTA]. After incubation at 4°C for 10 min, the lysates were centrifuged at 13,000 rpm in a microfuge for 30 min and the supernatant was collected (nuclear fraction). The protein concentrations in the cytoplasmic and nuclear fractions were then determined. The quality of the fractionation was assessed by immunoblotting with histone H3 and tubulin.

Antibodies and immunologic methods. Immunoblotting and immunoprecipitation were done as described (36). The intensities of signals on immunoblots were quantified with ImageJ software (NIH) using appropriate serial dilution of the samples as calibration. Indirect immunofluorescence microscopy was done as previously described (37). TRITC- and FITC-conjugated secondary antibodies were from DAKO (Glostrup, Denmark). Rabbit polyclonal antibodies against FLAG tag (29) and monoclonal antibodies against FLAG tag (M2; ref. 31), HA tag (12CA5; ef. 29), and tubulin (YL1/2; ref. 38) were obtained from sources as previously described. Monoclonal antibodies against MDM2 (2A10; Calbiochem, San Diego, CA), myc tag (9E10; DAKO), V5 tag (R960-25; Invitrogen), and p53 (DO1; Santa Cruz Biotechnology, Santa Cruz, CA) and polyclonal antibodies against p21CIP1/WAF1 (sc-397; Santa Cruz Biotechnology) were obtained from the indicated sources.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
{Delta}p53 is not active as a transcriptional factor for CIP1/WAF1 and MDM2. Given that {Delta}p53 lacks a portion of the DNA binding domain (Fig. 1A), we first compared the transcriptional activity of {Delta}p53 with those of other DNA binding–defective mutants of p53. The various constructs of wild-type and mutant p53 used in this study are shown in Fig. 1A. Compared with the control reaction, ectopic expression of wild-type p53 in H1299 cells (a p53-null cell line) induced the p53-responsive gene products p21CIP1/WAF1 and MDM2 (or HDM2; Fig. 1B). As expected, neither R273H nor R249S (both are missense "hotspot" mutants found in a variety of tumors) stimulated the expression of the same p53-responsive products. We found that even when expressed to the similar levels as p53, {Delta}p53 did not activate the endogenous p21CIP1/WAF1 or MDM2.

To further validate that {Delta}p53 did not possess transcriptional activity, luciferase reporters under the control of p21CIP1/WAF1 or MDM2 promoters were coexpressed with {Delta}p53 (Fig. 1B). The luciferase activities were normalized with the Renilla luciferase activity from a cotransfected plasmid to correct for transfection efficiency. As expected, both p21CIP1/WAF1 and MDM2 promoters were robustly transactivated by wild-type p53. In contrast, neither the DNA binding–defective mutants (R273H and R249S) nor {Delta}p53 significantly transactivated the promoters. These data indicate that ectopically expressed {Delta}p53 does not display intrinsic transcriptional activity toward endogenous or cotransfected p21CIP1/WAF1 and MDM2 promoters.

{Delta}p53 can bind to p53 but does not inhibit the transcriptional activity of p53. Because {Delta}p53 did not possess transcriptional activity, it is conceivable that it can act in a dominant-negative manner by virtue of its tetramerization with full-length p53. To test this hypothesis, we first examined if {Delta}p53 could indeed form a complex with p53. FLAG-tagged {Delta}p53 was coexpressed with HA-tagged p53 and was immunoprecipitated using a FLAG antiserum. Figure 1C shows that HA-p53 was coimmunoprecipitated with FLAG-{Delta}p53 but not with the control serum, confirming that {Delta}p53 could form a complex with p53.

To determine if the activity of p53 could be altered by {Delta}p53, a constant amount of p53-expressing plasmids was cotransfected with increasing amount of {Delta}p53-expressing plasmids (Fig. 1D). As both p53 and {Delta}p53 were FLAG tagged and of different sizes, their relative levels could be assessed by immunoblotting for FLAG. In agreement with the above data, p53 but not {Delta}p53 alone induced the expression of p21CIP1/WAF1 and MDM2. Unexpectedly, {Delta}p53 did not suppress the expression of p21CIP1/WAF1 and MDM2 induced by p53. We instead observed a slight increase in MDM2 expression when {Delta}p53 was coexpressed with p53.

To validate that {Delta}p53 was inadequate in reducing the activity of p53, a p21CIP1/WAF1 promoter-luciferase reporter was cotransfected with p53 and {Delta}p53. Figure 2A shows that the p21CIP1/WAF1 promoter was activated by p53, but it was not hindered in the presence of {Delta}p53. Because both wild-type p53 and {Delta}p53 were detected together with the same monoclonal antibody on the same blot, their relative level could be quantified by densitometry with the appropriate serial dilution standards. Given that this approach depended only on the relative expression between p53 and {Delta}p53, data from several independent experiments could be pooled. Figure 2A summarizes the experiments that examined the effects of {Delta}p53 on p53.


Figure 2
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  		Figure 2. {Delta}p53 can form complexes with p53 but does not affect the transcriptional activity of p53. A, transactivation of p21CIP1/WAF1 promoter by p53 is not affected by {Delta}p53. Cells were transfected with plasmids expressing a p21CIP1/WAF1 promoter-luciferase reporter and Renilla luciferase. Constant amount of FLAG-p53 and varying amounts of FLAG-{Delta}p53 were transfected as indicated (top). Cell extracts were prepared and the luciferase activities were determined. The transcriptional activity was expressed as a percentage of p53 alone (lane 2). The expressions of p53 and {Delta}p53 were detected together by immunoblotting for FLAG. Data from several experiments were pooled to construct the inhibition curve of {Delta}p53 on p53 activity (bottom). Dotted lines, theoretical inhibition curves as previously described (26). The various curves are based on the assumption that tetramers are only active with the number of wild-type p53 ranging from four to one. 4W, 4; 3W, ≥3; 2W, ≥2; 1W, ≥1. B, transactivation of MDM2 promoter by p53 is not impaired by {Delta}p53. Experiments were done as in (A) except that an MDM2 promoter-luciferase reporter was used. C, {Delta}p53 does not suppress cyclin B1 promoter. Cells were transfected with plasmids expressing a cyclin B1 promoter-luciferase reporter and Renilla luciferase. FLAG-tagged p53 or {Delta}p53 was coexpressed as indicated. The cells were harvested at 24 h after transfection and cell lysates were prepared. The luciferase activities were determined, normalized with the Renilla luciferase activity, and plotted as a percentage of control. The expression of FLAG-tagged p53 and {Delta}p53 was confirmed by immunoblotting. D, {Delta}p53 does not impair the suppression of the cyclin B1 promoter by p53. Experiments were done as in (A) except that a cyclin B1 promoter-luciferase reporter was used.

 
The dominant-negative activity of p53 mutants depends on the number of mutant molecules needed to be present in a tetramer to inhibit the activity of whole tetramer. We have previously made theoretical predictions of the inhibition characteristics of p53 DNA binding–defective mutants when the concentration of mutant is increased relative to the wild-type (26). The various inhibition curves, based on the assumption that tetramers are only active with the number of wild-type subunit required for full activity ranging from four (4W) to only one (1W), are also plotted in Fig. 2A. Our data revealed that {Delta}p53 did not inhibit p53 even when {Delta}p53 was expressed at a concentration much higher than p53. Similarly, we found that {Delta}p53 was equally ineffective in suppressing the activity of p53 on the MDM2 promoter (Fig. 2B).

Apart from transcription activation, p53 also represses the transcription of several genes like cyclin B1 (39). When expressed on its own, {Delta}p53 did not significantly reduce the expression from a cyclin B1-promoter reporter construct (Fig. 2C). As a control, wild-type p53 was able to suppress the cyclin B1 promoter (lane 2). Furthermore, coexpression of {Delta}p53 did not affect the p53-mediated repression of cyclin B1 promoter (Fig. 2D). Taken together, these data indicate that although {Delta}p53 can form a complex with p53, it is ineffective in inhibiting the activity of p53.

{Delta}p53 lacks the major NLS and is mainly localized to the cytoplasm. Nuclear localization of p53 is mediated by a NLS situating between the DNA binding domain and the tetramerization domain (40). This NLS (residues 305–321; see Fig. 1A) is notably absent in {Delta}p53. To examine the subcellular localization of {Delta}p53, epitope-tagged p53 or {Delta}p53 was expressed in H1299 cells and their localization was detected by indirect immunofluorescence microscopy (Fig. 3A ). As expected, both p53 and R273H mutant were predominantly localized to the nucleus. In marked contrast, {Delta}p53 was excluded from the nucleus and accumulated in the cytoplasm.


Figure 3
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  		Figure 3. {Delta}p53 is localized to the cytoplasm and can influence the localization of full-length p53. A, {Delta}p53 is localized to the cytoplasm. H1299 cells were transfected with plasmids expressing FLAG-tagged p53, R273H, or {Delta}p53 as indicated. At 24 h after transfection, the cells were fixed and the FLAG-tagged proteins were detected by immunostaining with a monoclonal antibody against FLAG, followed by a TRITC-conjugated antimouse immunoglobulin G (IgG) secondary antibody (red). Nuclei were counterstained with Hoechst 33258 (blue). Right, merged images. B, {Delta}p53 and p53 mutually affect each other's subcellular localization. Cells were cotransfected with plasmids expressing untagged p53 and FLAG-tagged {Delta}p53 (top) or FLAG-tagged p53 and V5-tagged {Delta}p53 (bottom). At 24 h after transfection, the cells were fixed and subjected to immunostaining. FLAG-{Delta}p53 was detected with a monoclonal antibody against FLAG, followed by a TRITC-conjugated antimouse IgG secondary antibody (red). FLAG-p53 was detected with a polyclonal antibody against FLAG, followed by a TRITC-conjugated antirabbit IgG secondary antibody (red). V5-{Delta}p53 was detected with a monoclonal antibody against V5, followed by a FITC-conjugated antimouse IgG secondary antibody (green). Nuclei were counterstained with Hoechst 33258 (blue). Right, merged images. C, confirmation of the cytoplasmic localization {Delta}p53 by subcellular fractionation. H1299 cells were transfected with plasmids expressing FLAG-tagged p53 and {Delta}p53 as indicated. At 24 h after transfection, the cells were either mock treated or irradiated with 50 J/m2 UVB. After incubation for 6 h, the cells were harvested and subjected to subcellular fractionation. The abundance of p53 and {Delta}p53 in total lysates, cytoplasmic fractions, and nuclear fractions was detected by immunoblotting for the FLAG tag.

 
Because {Delta}p53 and p53 were individually localized to distinct compartments but could form a complex when coexpressed, this prompted us to explore the localization of {Delta}p53 and p53 when they were coexpressed. When {Delta}p53 (FLAG tagged) was coexpressed with full-length p53 (untagged), many cells displayed a prominent nuclear staining of {Delta}p53. Representational images are shown in Fig. 3B. To detect both p53 and {Delta}p53 simultaneously in the same cells, {Delta}p53 and p53 were engineered to fuse with V5 and FLAG tags, respectively. Similar to FLAG-{Delta}p53, V5-{Delta}p53 was exclusively localized to the cytoplasm when expressed on its own (data not shown), but a portion was redistributed to the nucleus when coexpressed with FLAG-p53 (Fig. 3B). Conversely, {Delta}p53 caused a slight increase of FLAG-p53 in the cytoplasm. The effect of {Delta}p53 on p53 was not as profound as the converse, as many cells still retained a predominantly nuclear staining of p53.

The cytoplasmic localization of {Delta}p53 was further confirmed by subcellular fractionation. Figure 3C shows that, in marked contrast to p53, {Delta}p53 was predominantly present in the cytoplasmic fractions. There was a slight increase of {Delta}p53 in the nucleus when it was coexpressed with p53. In addition, we found that the localization of {Delta}p53 was not altered after DNA damage induced by UVB.

As a further control, we constructed a COOH-terminally deleted mutant (C{Delta}257) that removed the tetramerization domain from {Delta}p53 (Fig. 1A). As expected, C{Delta}257 did not possess transcriptional activity on p21CIP1/WAF1 promoter or MDM2 promoter (Fig. 4A ). Moreover, C{Delta}257 did not diminish the transactivation of p21CIP1/WAF1/MDM2 promoters by wild-type p53. Significantly, C{Delta}257 (which was exclusively cytoplasmic) did not affect the localization of p53, or vice versa (Fig. 4B).


Figure 4
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  		Figure 4. {Delta}p53 is targeted to ubiquitination-dependent degradation, and {Delta}p53 lacking the COOH-terminal tetramerization domain does not affect the activity or localization of p53. A, C{Delta}257 does not inhibit the transactivation of p21CIP1/WAF1 promoter by p53. Cells were transfected with plasmids expressing Renilla luciferase and p21CIP1/WAF1 promoter (left) or MDM2 promoter (right) luciferase reporters. FLAG-tagged p53 and C{Delta}257 were expressed as indicated. Cell extracts were prepared and luciferase activities were determined (bottom). The transcriptional activity was expressed as a percentage of p53 alone (lane 2). The expression of FLAG-tagged p53 and C{Delta}257 was confirmed by immunoblotting. B, C{Delta}257 is localized to the cytoplasm and does not affect the nuclear localization of p53. H1299 cells were transfected with plasmids expressing FLAG-tagged C{Delta}257 and V5-p53. At 24 h after transfection, the cells were fixed and subjected to immunostaining. V5-p53 was detected with a monoclonal antibody against V5, followed by a FITC-conjugated antimouse IgG secondary antibody (green). FLAG-C{Delta}257 was detected with a polyclonal antibody against FLAG, followed by a TRITC-conjugated antirabbit IgG secondary antibody (red). Nuclei were counterstained with Hoechst 33258 (blue). Right, merged images. C, {Delta}p53 is targeted to MDM2-dependent ubiquitination. FLAG-tagged p53 or {Delta}p53 was coexpressed with MDM2 and HA-ubiquitin (Ub) in H1299 cells. The cells were treated with the proteasome inhibitor LLnL for 6 h before harvest to stabilize the ubiquitinated products. Cell extracts were prepared and subjected to immunoprecipitation with either control normal rabbit serum or FLAG antiserum. The immunoprecipitates were immunoblotted with antibodies against HA, FLAG, and MDM2 as indicated. The positions of unmodified and polyubiquitinated p53 are indicated. Left, positions of molecular size standards (in kilodaltons). D, {Delta}p53 is less stable than wild-type p53. FLAG-tagged p53, {Delta}p53, or {Delta}p53-NLS was coexpressed with MDM2 in H1299 cells. At 48 h after transfection, doxycycline and cycloheximide were added and cell extracts were prepared at the indicated time points. The stability of the FLAG-tagged proteins was examined by immunoblotting.

 
Collectively, these results indicate that, unlike full-length p53, {Delta}p53 is not imported into the nucleus. This may explain, in part, why {Delta}p53 is not active as a transcription factor and is not an inhibitor of p53.

{Delta}p53 is subjected to ubiquitination and is less stable than wild-type p53. A major pathway of p53 proteolysis involves MDM2-mediated ubiquitination. MDM2 binds to the NH2-terminal region of p53, shuttles p53 to the cytoplasm, and targets p53 for ubiquitination. Ubiquitinated p53 is then degraded by the proteasome complex. Ubiquitination occurs at lysine residues at the COOH-terminal region and at the NH2-terminal half of the DNA binding domain (ref. 41 and references therein). Given that {Delta}p53 still retains the NH2-terminal MDM2 binding site, as well as the potential ubiquitin-acceptor sites, we next investigated if {Delta}p53 is subjected to ubiquitination. Cell-free extracts were prepared from cells expressing FLAG-tagged p53 or {Delta}p53 together with MDM2 and HA-ubiquitin. The FLAG-tagged proteins were immunoprecipitated and the ubiquitinated proteins were detected by immunoblotting for HA (Fig. 4C). As expected, high molecular size products representing ubiquitinated proteins could be detected in the FLAG-p53 immunoprecipitates but not in the control serum immunoprecipitates. Likewise, high molecular size products containing HA-ubiquitin were readily detected in the immunoprecipitates of FLAG-{Delta}p53, indicating that {Delta}p53 was ubiquitinated. Finally, MDM2 bound specifically with p53 and {Delta}p53 but not with the control serum immunoprecipitates.

Given that, unlike p53, {Delta}p53 is already in the cytoplasm and does not require a nuclear exporting step for degradation, it is conceivable that {Delta}p53 is degraded more efficiently than p53. To test this hypothesis, cells expressing p53 or {Delta}p53 were treated with doxycycline (the promoters of these constructs were under the negative control of doxycycline) and cycloheximide. As we have previously shown with the same assay (41), ectopically expressed p53 was degraded relatively slowly (Fig. 4D). In contrast, a similar expression level of {Delta}p53 was degraded quicker than p53.

Taken together, these data indicate that, similar to full-length p53, {Delta}p53 can bind MDM2 and be targeted for ubiquitination. Moreover, {Delta}p53 exhibits a shorter half-life than p53, probably due, in part, to its cytoplasmic localization.

Forcing {Delta}p53 into the nucleus potentiates its dominant-negative activity. To test if the relatively short half-life of {Delta}p53 was due to its cytoplasmic localization, three NLS (as well as a myc tag) were added to the COOH terminus of {Delta}p53 (Fig. 1A). As expected, reinstating NLS to {Delta}p53 restored the nuclear localization (Fig. 5A ). In contrast to {Delta}p53, {Delta}p53-NLS was degraded at a similar rate as wild-type p53 (Fig. 4D), suggesting that the instability of {Delta}p53 may be due, in part, to its cytoplasmic localization.


Figure 5
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  		Figure 5. NLS-containing {Delta}p53 does not possess transcriptional activity and is a weak dominant-negative protein. A, {Delta}p53-NLS does not affect the localization of p53. Cells were transfected with plasmids expressing FLAG-tagged p53 and myc-tagged {Delta}p53-NLS. At 24 h after transfection, the cells were fixed and subjected to immunostaining. FLAG-p53 was detected with a polyclonal antibody against FLAG, followed by a TRITC-conjugated antirabbit IgG secondary antibody (red). {Delta}p53-NLS was detected with a monoclonal antibody against myc, followed by a FITC-conjugated antimouse IgG secondary antibody (green). Nuclei were counterstained with Hoechst 33258 (blue). Right, merged images. B, {Delta}p53-NLS can form a complex with p53. HA-tagged p53 was coexpressed with FLAG-{Delta}p53 or FLAG-{Delta}p53-NLS. Cell extracts were prepared and 100 µg were subjected to immunoprecipitation with either control normal rabbit serum or FLAG antiserum as indicated. The immunoprecipitates were immunoblotted for HA and FLAG. Total cell lysates (10 µg) were applied to indicate the input. C, MDM2 and p21CIP1/WAF1 are not activated by {Delta}p53-NLS. An MDM2 promoter-luciferase reporter and a Renilla luciferase construct were coexpressed with p53, {Delta}p53, or {Delta}p53-NLS as indicated. Cell extracts were prepared at 24 h after transfection and the abundance of endogenous p21CIP1/WAF1, MDM2, and recombinant p53 proteins was detected by immunoblotting (top). Equal loading of lysates was confirmed by immunoblotting for tubulin. The luciferase activity was determined, normalized with the Renilla luciferase activity to correct for variations in transfection efficiency between samples, and plotted as a percentage of p53 (bottom). Columns, mean of three independent experiments; bars, SD. D, effects of {Delta}p53-NLS on the activity of p53. Constant amount of FLAG-p53 and increasing amount of {Delta}p53-NLS were expressed in H1299 cells as indicated. At 24 h after transfection, cell extracts were prepared and the abundance of p21CIP1/WAF1 and MDM2 was detected by immunoblotting. The expression of p53 and {Delta}p53-NLS was confirmed by immunoblotting. Tubulin analysis was included to assess protein loading and transfer.

 
It is possible that the relatively weak dominant-negative action of {Delta}p53 on p53 was also due to its subcellular localization. To test this hypothesis, we examined if {Delta}p53-NLS could modulate the transcriptional activity of p53. One of our concerns was that the addition of the three NLS and myc tag at the COOH terminus might affect the function of the nearby tetramerization domain. Immunoprecipitation revealed that, similar to {Delta}p53, {Delta}p53-NLS was able to form a complex with p53 (Fig. 5B), validating that the extra NLS targeted {Delta}p53 to the nucleus without affecting oligomerization. We next examined the subcellular localization of p53 and {Delta}p53-NLS by immunostaining. In marked contrast to {Delta}p53, {Delta}p53-NLS did not affect the nuclear localization of p53 (Fig. 5A).

Interestingly, although {Delta}p53-NLS was localized to the nucleus, it was unable to induce the expression of p21CIP1/WAF1 or MDM2 (Fig. 5C). This was further validated by the lack of transactivation activity of {Delta}p53-NLS on a cotransfected MDM2 promoter (Fig. 5C). These data unequivocally show that the lack of intrinsic transcriptional activity of {Delta}p53 was not simply due to deficiency of NLS but was likely to be due to the incomplete DNA binding domain.

Because nuclear-targeting {Delta}p53 did not possess transcriptional activity, it is possible that it could act in a dominant-negative fashion. To test this hypothesis, {Delta}p53-NLS was coexpressed with wild-type p53 and the transcriptional activity was measured. Figures 5D and 6A show that the activities of p53 were only marginally reduced by {Delta}p53-NLS. The effect was slightly more prominent at high doses of {Delta}p53-NLS. Consistent with the effects of {Delta}p53 (Fig. 1D), endogenous MDM2 expression was actually stimulated by lower doses of {Delta}p53-NLS (Fig. 5D). The inhibitory activity of {Delta}p53-NLS seemed to be slightly stronger than that of {Delta}p53 (Fig. 6A) and is comparable to other DNA binding–defective mutants (3). To obtain more definite evidence of the importance of nuclear localization in the dominant-negative action of p53, we also removed the entire NH2-terminal region in the {Delta}p53 backbone (N{Delta}159). The basis of this is that NH2-terminally truncated versions of p53 are more powerful inhibitors of p53 functions than DNA binding–defective mutants (3). Figure 6B shows that the transcriptional activity of p53 was effectively attenuated by {Delta}p53N{Delta}-NLS. Moreover, the expression of endogenous p21CIP1/WAF1 and MDM2 was also down-regulated by {Delta}p53N{Delta}-NLS (Fig. 6C). Significantly, {Delta}p53N{Delta}-NLS inhibited p53 function better than {Delta}p53N{Delta} (without NLS), indicating a critical role of nuclear localization in the dominant-negative function of p53 (Fig. 6D).


Figure 6
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  		Figure 6. The dominant-negative activity of {Delta}p53 lacking the NH2-terminal region is increased by nuclear localization. A, {Delta}p53-NLS only slightly reduces the transcriptional activity of p53. H1299 cells were transfected with plasmids expressing a MDM2 promoter-luciferase reporter, p53, and FLAG-tagged {Delta}p53-NLS as indicated. Cell extracts were prepared and the luciferase activities were determined. The transcriptional activity was expressed as a percentage of p53 alone (lane 2). The expression of p53 and {Delta}p53-NLS was confirmed by immunoblotting. Data from several experiments were pooled to construct the inhibition curve of FLAG-{Delta}p53 ({circ}) or FLAG-{Delta}p53-NLS (bullet) on p53 activity (bottom). The transcriptional activity was plotted against the ratio of {Delta}p53/p53 or {Delta}p53-NLS/p53 as described in Fig. 2A. B, {Delta}p53N{Delta}-NLS strongly inhibits the transcriptional activity of p53. H1299 cells were transfected with plasmids expressing a MDM2 promoter-luciferase reporter, FLAG-p53, and FLAG-{Delta}p53N{Delta}-NLS as indicated. Cell extracts were prepared and the luciferase activities were determined (bottom). The transcriptional activity was expressed as a percentage of p53 alone (lane 2). The expression of p53 and {Delta}p53N{Delta}-NLS was confirmed by immunoblotting. C, {Delta}p53N{Delta}-NLS decreases the transactivation of p21CIP1/WAF1 and MDM2 by p53. Constant amount of FLAG-p53 and increasing amount of FLAG-{Delta}p53N{Delta}-NLS were expressed in H1299 cells as indicated. At 24 h after transfection, cell extracts were prepared, and the abundance of p21CIP1/WAF1 and MDM2 was detected by immunoblotting. The expression of p53 and {Delta}p53N{Delta}-NLS was confirmed by immunoblotting for FLAG. Tubulin analysis was included to assess protein loading and transfer. D, {Delta}p53N{Delta}-NLS is a more potent inhibitor of p53 than {Delta}p53N{Delta}. H1299 cells were transfected with plasmids expressing a MDM2 promoter-luciferase reporter and different ratios of plasmids expressing FLAG-tagged p53 and {Delta}p53N{Delta} ({circ}) or {Delta}p53N{Delta}-NLS (bullet). The transcriptional activity was plotted against the ratio of {Delta}p53N{Delta}/p53 or {Delta}p53N{Delta}-NLS/p53 as described in Fig. 2A.

 
Taken together, these data show that {Delta}p53 remains inactive even when nuclear localization is restored. Targeting {Delta}p53 to the nucleus does enhance its dominant-negative activity, thus explaining why {Delta}p53 is not a robust dominant-negative protein.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It is remarkable that whereas p53 is one of the most investigated human genes, it has only recently been recognized that it has the potential to encode a large number of isoforms. Whereas the existence of {Delta}p53 has been subjected to some debates (17), the mechanistic insights drawn from the study of novel forms of this critical tumor suppressor can be revealing. Similar to DNA binding–defective mutants like R249S and R273H, {Delta}p53 did not display intrinsic transcriptional activity. We found that {Delta}p53 activated neither endogenous p21CIP1/WAF1 nor MDM2 (Fig. 1B). Likewise, cotransfected p21CIP1/WAF1 or MDM2 promoters were not transactivated by {Delta}p53 (Fig. 1B). In addition, unlike wild-type p53, {Delta}p53 failed to repress the cyclin B1 promoter. Hence, it is paradoxical that {Delta}p53 was reported to display activity, in particular after DNA damage during S phase (25). The molecular mechanism underlying this activity remains to be elucidated. It is conceivable that posttranslational modifications triggered during S phase or after DNA damage may contribute to the activation of {Delta}p53. Here, we mainly compared the intrinsic transcriptional activities between transiently transfected p53 and {Delta}p53 without additional stress. However, we were also not able to detect significant p21CIP1/WAF1 transcriptional activity1 or a change in subcellular localization (Fig. 3C) with transfected {Delta}p53 after UV irradiation (cells were irradiated with 50 J/m2 UVB and harvested after 6 h). As Rohaly et al. (25) used stable H1299 cell lines that conditionally expressed {Delta}p53, a possibility is that additional mutations in the cell lines may contribute to the activity of {Delta}p53.

Two factors may account for the inactivity of {Delta}p53. First, the COOH-terminal 35 residues of the DNA binding domain are absent in {Delta}p53. As mutation of single residues in this region (e.g., R273H) is sufficient to disrupt the transcriptional activity of p53, it is a fair postulation that {Delta}257–322 may be detrimental to the structure of the DNA binding domain. Another factor that may contribute to the inactivity of {Delta}p53 is that it is not imported into the nucleus. Nuclear localization of p53 is mediated by a major NLS (absent in {Delta}p53) and two minor NLS at the COOH-terminal region (40). Indeed, we found that ectopically expressed {Delta}p53 was excluded from the nucleus (Fig. 3A and C). Addition of leptomycin B, a CRM1 inhibitor, did not affect the localization of {Delta}p53.1 These observations indicate that the cytoplasmic localization of {Delta}p53 is due to a defect in nuclear import and not due to a more active nuclear export in comparison with p53.

To see if the inactivity of {Delta}p53 can be explained entirely by its cytoplasmic localization, we constructed a version of {Delta}p53 containing three NLS from the SV40 large T antigen. However, although {Delta}p53-NLS was correctly localized to the nucleus, it did not activate p21CIP1/WAF1 or MDM2 (Fig. 5C). These results indicate that the lack of an effective NLS in {Delta}p53 is not the main reason for the absence of transcriptional activity and underscore the critical role of the DNA binding domain.

The localization of {Delta}p53 to the cytoplasm does seem to contribute to the relative instability of the protein. We showed that, similar to full-length p53, {Delta}p53 was ubiquitinated in the presence of MDM2 (Fig. 4C). This is not too surprising as {Delta}p53 still retains the NH2-terminal MDM2 binding site. Indeed, we found that MDM2 was coimmunoprecipitated with {Delta}p53 (Fig. 4C). Furthermore, the potential ubiquitin-acceptor sites are retained in {Delta}p53. Although a cluster of six lysine residues found in p53 are absent in {Delta}p53, we have shown that these lysine residues are not critical ubiquitination sites (41). Instead, both the NH2-terminal and COOH-terminal clusters of ubiquitination acceptor sites are still present in {Delta}p53. Moreover, there is a high degree of flexibility in the sites of ubiquitination, so the sequence missing in {Delta}p53 is unlikely to impair the overall ubiquitination of the protein.

Although {Delta}p53 seemed to be more efficiently ubiquitinated than full-length p53 (Fig. 4C), the ubiquitination assays were not quantitative and we were not able to unequivocally conclude that {Delta}p53 is more susceptible to ubiquitination than p53. Analysis of the stability of the proteins revealed that {Delta}p53 was less stable than full-length p53 (Fig. 4D). We attribute this difference of the half-lives mainly to the subcellular localization of {Delta}p53 and p53. Although p53 can be ubiquitinated by MDM2 inside the nucleus (42), one major pathway of p53 degradation is through the export of the p53-MDM2 complexes to the cytoplasm before p53 is delivered to the ubiquitin/proteasome pathway. As {Delta}p53 is already in the cytoplasm, it is possible that it can be degraded by the ubiquitin/proteasome pathway more efficiently. In support of this, we found that {Delta}p53-NLS, which was imported into the nucleus, was more stable than {Delta}p53 (Fig. 4D). Because ubiquitination itself also contributes to the efficient export of p53 to the cytoplasm (43, 44), it could be hypothesized that the two events, ubiquitination and export, simply act reciprocally on each other for {Delta}p53. We think that this is unlikely because treatment with leptomycin B did not increase the nuclear localization of {Delta}p53,1 suggesting that {Delta}p53 was never imported into the nucleus in the first place.

Another consequence of the cytoplasmic localization of {Delta}p53 is the lack of dominant-negative activity. We found that the transcriptional activities of p53 (including the activation of p21CIP1/WAF1 and MDM2 promoters as well as the repression of the cyclin B1 promoter) were not significantly affected by {Delta}p53 (Fig. 2). It is interesting that the activation of MDM2 by p53 was actually increased by {Delta}p53 (Figs. 1D and 5D). This is also consistent with the increase of p53 activity on the MDM2 promoter in the presence of the R273H mutant (26). Because the presence of one or two molecules of {Delta}p53 (or R273H) within a tetramer may not be inhibitory, the addition of {Delta}p53 (up to certain level) may in fact increase the abundance of active tetramers.

Although {Delta}p53 did not strongly inactivate p53, the localization of p53 was nevertheless altered because some p53 staining could be detected in the cytoplasm (Fig. 3B). We postulate that this was due to the complex formation between {Delta}p53 and p53, rendering a portion of p53 to be imported into the nucleus less efficiently. This impeded nuclear accumulation was apparently not sufficient to reduce the activity of p53, possibly because the majority of p53 was still imported into the nucleus. It is not too surprising as nuclear import is an active process and does not depend on tetramerization (COOH-terminally truncated p53 lacking the tetramerization domain is still imported; ref. 26).

Conversely, a portion of {Delta}p53 was imported into the nucleus when it was coexpressed with p53 (Fig. 3B and C). This was presumably again due to the interaction between p53 and {Delta}p53, with {Delta}p53 piggybacked into the nucleus. Indeed, some proteins without NLS are imported into the nucleus by a similar principle. For example, cyclin D1-CDK4 complexes are targeted to the nucleus by binding to NLS-containing CDK inhibitors (45, 46). This increase in nuclear {Delta}p53 was insufficient to inhibit p53, presumably because of the relatively low levels of {Delta}p53 in the nucleus. Another reason is that even when {Delta}p53 is imported into the nucleus, several copies per tetramer are probably required to abolish the transcriptional activity. This was verified by the experiments involving {Delta}p53-NLS (Fig. 6A). Although {Delta}p53-NLS was localized to the nucleus and could bind p53, it displayed an inhibitory profile similar to the theoretical prediction that at least three subunits are required to inhibit the tetramer. This is similar to the activity displayed by other DNA binding–defective mutants like R273H and R249S (26).

To obtain a clearer indication of the effect of nuclear localization on {Delta}p53, we accentuated the dominant-negative effect of {Delta}p53 by removing its NH2-terminal region. In marked contrast to DNA binding–defective mutants, N{Delta} mutants are powerful inhibitors of p53 function and about one mutant per tetramer is sufficient to abolish the transcriptional activity (26). Consistent with this, we found that {Delta}p53N{Delta}-NLS was a more robust inhibitor of p53 than {Delta}p53-NLS (Figs. 6B and C). Furthermore, {Delta}p53N{Delta}-NLS inhibited p53 more efficiently than {Delta}p53N{Delta} (Fig. 6D), indicating that the nuclear localization is critical for the dominant-negative function of p53.


   Acknowledgments
 
Grant support: Research Grants Council grants HKUST6439/06M and HKUST6123/04M (R.Y.C. Poon).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Sandy Siu and Anita Lau for technical assistance and members of the Poon laboratory for constructive criticism of this study.


   Footnotes
 
1 Our unpublished data. Back

Received 9/28/06. Revised 12/ 6/06. Accepted 12/20/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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