p53CP is p51/p63, the third member of the p53 gene family: partial purification and characterization
Mingjia Tan,
Junhui Bian2,
Kunliang Guan1 and
Yi Sun3
Department of Molecular Biology, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, MI 48105, USA and
1 Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48019, USA
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Abstract
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The p53 tumor suppressor is a transcription factor that upon activation by DNA-damaging agents induces growth arrest or apoptosis mainly through transactivation and transrepression of its downstream target genes. Two additional p53 family members, p73 and p51/p63, were recently identified and characterized. Although the three family members share some similarities in transcription activation and apoptosis induction, each of them appears to play a distinct role in development and tumor suppression. We have previously identified a nuclear protein, p53CP (p53 competing protein), that is not p53 but binds to the p53 consensus sequence. Here we report the partial purification of p53CP from HeLa cells by ammonium sulfate precipitation, followed by a series of chromatography steps through heparinagarose, Mono S ion exchange and DNA affinity columns, coupled with a gel shift assay. Although p53CP activity is readily detectable in HeLa cells by gel shift assay, only a trace amount of p53CP protein was partially purified, which was not sufficient for direct protein sequencing. Using a monoclonal antibody (4A4) specific for all p51/p63 isoforms or a polyclonal antibody (N-18) recognizing the N-terminus-containing p51/p63 isoforms we detected a significant enrichment of p51/p63 protein in p53CP-containing fractions following each step of purification. Significantly, p51/p63 was detected only in the DNA affinity column fractions that contain p53CP activity. Thus, p53CP appears to be p51/p63, the third member of the p53 gene family.
Abbreviations: DTT, dithiothreitol; NPC, nasopharyngeal carcinomas; PMSF, phenylmethylsulfonyl fluoride.
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Introduction
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The p53 tumor suppressor, a 53 kDa nuclear protein, is a transcription factor that is activated through phosphorylation and acetylation in response to environmental stimuli such as DNA damage, hypoxia, redox disturbance and oncogene activation (1,2). Activated p53 induces growth arrest to ensure that damaged DNA is repaired in cells before re-entering the cell cycle or induces apoptotic cell death to eliminate cells when repair is impossible (3). Thus, p53 serves as guardian of the genome to prevent gene amplification and maintain the genetic integrity of the cells (4). Structurally, p53 protein consists mainly of three distinct domains: a transactivation domain at the N-terminus, a central specific DNA-binding domain and a tetramerization and regulatory domain at the C-terminus of the molecule (5). As a transcription factor, p53 specifically binds to its consensus DNA binding sequence, consisting of two repeats of the 10 bp motif 5'-PuPuPuC (A/T)(T/A)GPyPyPy-3' separated by 013 bp (6), and transactivates expression of many target genes (7). Growth arrest induced by p53 is mainly mediated by Waf-1/p21 (8), 14-3-3
(9) and PTGF-ß (10). Although the mechanism by which p53 induces apoptosis is less well understood, it appears to involve activation of p53 target genes including Bax (11), KILLER/DR5 (12), Fas/APO1 (11), PAG608 (13) and the redox-related PIG and GPX genes (14,15). p53 was also found to regulate angiogenesis and metastasis via activation of other target genes (16). Activation of Mdm-2 by p53 serves as a negative auto-feedback loop to keep p53 levels in check (17). In addition, p53 was found to repress the expression of several genes, although the biological consequences of this inhibition are not well understood (18,19). Thus, through the transcriptional activation/repression of downstream target genes, p53 regulates several important cellular processes, including cell growth and differentiation, apoptosis, DNA repair/replication and angiogenesis (3). p53 mutations found in >50% of human cancers were clustered in the specific DNA-binding domain, resulting in p53 inactivation by abolishing p53-specific binding and transactivation (5).
Two additional p53 family members, p73 and p51/p63, were recently identified and characterized (2023). Like p53, both p73 and p51/p63 contain regions corresponding to the p53 N-terminal transactivation, central DNA-binding and C- terminal oligomerization domains (20,22). Due to their structural similarities, p73 and p51/p63 can bind to p53 consensus sequences, activate transcription of p53 target genes and induces apoptosis when overexpressed in cells (21,22,2427). Unlike p53, both p73 and p51/p63 have multiple splicing variants (28) and both contain a SAM-like domain at the C-terminus, known to be involved in proteinprotein interactions (29). Biologically, p73 and p51/p63 are more likely involved in neurogenesis (p73) and embryogenesis (p51/p63) rather than in cancer, as evidenced by mouse knockout studies (3033) and a mutational study that showed a very low frequency in human cancers (for a review see ref. 34). Thus, although three proteins belong to the same gene family, there are substantial differences in their normal physiological functions.
We have previously identified a nuclear protein, designated p53CP (p53 competing protein), that specifically binds to the consensus p53-binding sites found in several p53 downstream target genes (35). We have addressed the question of whether p53CP is p73 or p51/p63 or represents a new p53 family member. We report here the partial purification of p53CP from HeLa cells and provide evidence that p53CP is p51/p63, the third member of the p53 family.
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Materials and methods
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Purification of p53CP
The procedure used for purification of transcription factor Sp1 (36) was modified for p53CP purification as detailed below. A gel shift assay, detailed previously (35,37), was used to monitor fractions that contain p53CP activity.
Preparation of nuclear extracts
A sample of 100 l of HeLa S3 nuclear pellets was purchased from Cell Applications (San Diego, CA). The nuclear extract was prepared as described (38). Briefly, after being thawed on ice, the pellets were resuspended in 500 ml nuclear extract buffer [500 mM KCl, 25 mM HEPES, pH 7.8, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 20 µg/ml aprotinin and 0.1 mM dithiothreitol (DTT)] and slowly homogenized on ice. The sample was centrifuged at 22 000 g for 1 h and supernatants were collected and used as nuclear extract.
Ammonium sulfate precipitation
Ammonium sulfate was slowly added with gentle stirring to the nuclear extract up to a final concentration of 40%, followed by gentle stirring at 4°C for an additional 1 h. The mixture was centrifuged at 22 000 g for 30 min. The pellet was dissolved in TM buffer (50 mM TrisHCl, pH 7.8, 20 mM KCl, 10% glycerol, 1 mM PMSF, 10 µg/ml leupeptin, 20 µg/ml aprotinin and 0.1 mM DTT) and dialyzed against a large volume of TM buffer overnight. The dialyzate was centrifuged at 22 000 g to remove insoluble material. The protein concentration was measured with a Bio-Rad reagent kit. The supernatant was saved as fraction I and p53CP activity was examined by gel shift assay.
Heparinagarose chromatography
Fraction I was applied to an equilibrated heparinagrose column (25 ml bed volume) and p53CP was eluted with a linear gradient of KCl (0.11.0 M in TM buffer, pH 7.8) at a rate of 2 ml/min until the A280 dropped to 0. Alternate fractions were monitored by gel shift assay for p53CP activity and the fractions with activity were pooled and dialyzed against TM buffer at 4°C overnight. This fraction was saved as fraction II.
Ion exchange chromatography
A Mono S column was equilibrated with TM buffer for 2 h at 4°C. Fraction II was then loaded onto the equilibrated Mono S column (1 ml bed volume) and p53CP was eluted with a linear gradient of KCl (0.11.0 M in TM buffer, pH 7.8) at a rate 0.5 ml/min until the A280 dropped to 0. Each alternate fraction was measured by gel shift assay and the fractions containing p53CP activity were pooled and dialyzed against TM buffer overnight. This fraction was saved as fraction III.
DNA affinity column
The sequence-specific DNASepharose column was prepared as follows. To make unidirectional concatemers of T3SF (an oligonucleotide to which p53CP showed strong binding; ref. 35) the following complementary oligonucleotides with overhangs were used: 5'-GGGCTTGCTTGAACAGGGTC-3' and 5'-GCCCGACCCTGTTCAAGCAA-3'. The oligonucleotides (440 µg each) were mixed in 130 µl of TE buffer, boiled in a water bath for 5 min and then annealed overnight. The annealed oligonucleotides were phosphorylated at the 5'-end by incubation at 37°C for 2 h with a reaction mixture containing T4 nucleotide kinase (20 µl, 200 U), 20 mM ATP (30 µl) and 10x phosphorylation buffer (20 µl). The oligonucleotides were extracted with phenol/chloroform/isoamyl alcohol and ethanol precipitated. The resulting oligonucleotides were resuspended in 130 µl of H2O and ligated to form the concatemers by incubation at 15°C overnight with a reaction mixture containing 10x ligase buffer (20 µl), 20 mM ATP (40 µl) and T4 ligase (10 µl, 10 U). The concatemers (from dimer up to 20mers) were again extracted with phenol/chloroform, ethanol precipitated and resuspended in 100 µl of H2O. To conjugate the concatemers with Sepharose 4B, 3 g CNBr-activated Sepharose 4B was washed with 500 ml of 1 mM HCl, 100 ml of H2O and 100 ml of 10 mM potassium phosphate, pH 8.0, and then resuspended in 4 ml of 10 mM potassium phosphate, pH 8.0. Sepharose 4B was then incubated with 100 µl of concatemer oligonucelotides prepared as above at room temperature on a rotator overnight. The resin was washed with 100 ml of H2O twice and 100 ml of 1 M ethanolamine hydrochloride (pH 8.0) and then incubated with 5 ml of 1 M ethanolamine hydrochloride (pH 8.0) at room temperature for 4 h on a rotator. The final wash included 100 ml of the following solution sequentially: 10 mM potassium phosphate (pH 8.0), 1 M potassium phosphate (pH 8.0), 1 M KCl, H2O and column storage buffer (10 mM TrisHCl, pH 7.8, 1 mM EDTA, 0.3 M NaCl, 0.04% sodium azide). The resin was resuspended in 5 ml of column storage buffer at 4°C. For DNA affinity purification, fraction III was concentrated, mixed with poly(dIdC) to a final concentration of 1 µg/ml at 4°C for 10 min, to prevent non-specific binding, loaded onto the affinity column and eluted with a step gradient of KCl (0.051 M). Each fraction was monitored by gel shift assay for p53CP activity and SDSPAGE with silver staining for purity determination. For DNA affinity purification directly from nuclear extract, 510 ml of nuclear extract from a mouse liver tumor line (H-Tx) were dialyzed in cold binding buffer (20 mM TrisHCl, pH 7.5, 50 mM NaCl, 2.5 mM MgCl2, 0.5 mM DTT) at 4°C for 3 h in a Slide-A-Lyzer (Pierce) bag. Poly(dIdC) was added at 50 µg/ml. The resulting sample was passed through a T3SFSepharose 4B affinity column (0.5 ml volume) pre-equilibrated with binding buffer. After washing with 10 ml of binding buffer, a series of elutions were made with 0.5 ml of binding buffer containing various concentrations of NaCl (0.11 M). The presence of p53CP was monitored in subsequent gel shift assays using 2 µl of each fraction.
Sample preparation for protein sequencing
The fractions eluted with 0.3 and 0.4 M KCl, which contained p53CP activity, were combined, concentrated to a small volume (¬20 µl) with a Centricon dialysis apparatus and subjected to SDSPAGE, followed by Coomassie staining. Four faint bands with a size range of 4060 kDa were excised and subjected to microsequencing at the Department of Chemistry, PGRD.
Western blot analysis
The pooled fractions that contained p53CP after each step of purification or each concentrated fraction after DNA affinity chromatography directly of nuclear extract were subjected to western blot analysis as described (39). Antibodies used were a mouse monoclonal antibody 4A4 (SC-8431; Santa Cruz), raised specifically against all p51/p63 isoforms, and a goat polyclonal antibody N-18 (SC-8369; Santa Cruz), recognizing the N-terminus-containing p51/p63 isoforms.
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Results and discussion
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Purification of p53CP
We have previously identified a nuclear protein, p53CP, that has p53-like activity in that it binds to p53 consensus sequences found in several p53 target genes (35). To clone the gene encoding p53CP we have used 32P-labeled concatemerized T3SF oligonucleotide to screen several cDNA expression libraries (40), including a home-made, unamplified library from a mouse liver tumor line (H-Tx), the line in which p53CP was originally identified (35). We isolated several clones in the first two rounds of screening, but none of these positive clones was specific or could be enriched in a third cycle of screening (data not shown). Failure to clone the gene through this approach prompted us to use a conventional protein purification strategy coupled with protein microsequencing for gene cloning. The procedure for purification of p53CP was modified from Briggs et al. (36) and in each purification step p53CP activity was monitored by gel shift assay. Nuclear extract was prepared from 100 l of HeLa suspension culture, followed by ammonium sulfate precipitation. In a pilot experiment a small aliquot of nuclear extract was precipitated with ammonium sulfate at a final concentration of 20, 40 and 60%, followed by gel shift assay. It was found that maximum p53CP activity was present in the 40% ammonium sulfate precipitate (data not shown). This concentration was therefore used for large-scale purification.
Nuclear proteins (30 mg/ml) precipitated by ammonium sulfate (40%) were first separated on a heparinagrose column. After sample loading and pre-washing the proteins were eluted with a linear gradient of KCl (0.11 M in TM buffer, pH 7.8) at a rate of 2 ml/min, collecting 3 ml fractions. An aliquot of 8 µl of each alternate fraction collected was measured by gel shift assay to determine the presence of p53CP. As shown in Figure 1
, p53CP can be detected in nuclear extract and after ammonium sulfate precipitation. p53CP activity was detected in fractions 4854. These p53CP-containing fractions were pooled, dialyzed and loaded onto a Mono S column, eluted with a linear gradient of KCl (0.11.0 M in TM buffer, pH 7.8) at a rate of 0.5 ml/min, collecting 2 ml fractions. Again, 8 µl of each alternate fraction was assayed for p53CP. As shown in Figure 2
, fractions 4351, eluted by KCl concentrations between 0.315 and 0.625 M, contained p53CP. These fractions were pooled, dialyzed, concentrated with a Centrocon 10 apparatus and mixed with poly(dIdC). The mixture was then loaded onto a 2 ml DNA affinity column made of concatemerized T3SF. After washing, proteins were eluted with a step gradient of KCl (1 ml/fraction, 0.5 ml/tube at a concentration of 0.051 M). Again, an aliquot from each fraction was used for gel shift assay for p53CP and for SDSPAGE with silver staining for purity. As shown in Figure 3
, p53CP was detected in fractions 69 (mainly in fraction 8), corresponding to KCl concentrations of 0.30.4 M. Silver staining of each fraction showed co-purification of some other proteins (data not shown), indicating that our procedure led to partial purification of p53CP.

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Fig. 1. Purification of p53CP by heparinagarose chromatography. Nuclear extract was prepared from HeLa nuclear pellet, precipitated with 40% ammonium sulfate and loaded onto a heparinagarose column (bed volume 25 ml). The column was eluted with a linear gradient of KCl (0.11 M in TM buffer) at a rate of 2 ml/min. The fractions were collected and alternate fractions (8 µl) were measured for p53CP activity by gel shift assay. Arrow 1 indicates p53CP and arrow 2 points to a non-specific T3SF-binding protein, previously identified as a 40 kDa protein (35).
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Fig. 2. Purification of p53CP by Mono S ion exchange chromatography. The p53CP-containing fractions after heparinagarose purification were combined, dialyzed and loaded onto a Mono S ion exchange column (bed volume 1 ml), followed by linear gradient elution (0.11 M KCl in TM buffer) at a rate of 0.5 ml/min. Alternate fractions (8 µl) were used in a gel shift assay for p53CP activity.
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Fig. 3. Purification of p53CP by DNA affinity chromatography. The p53CP-containing fractions were pooled, dialyzed and concentrated before being loaded on a DNA affinity column made with concatemerized T3SF oligonucleotide. After washing, samples were eluted with 1 ml of an increasing concentration of KCl (0.051 M). The fractions were collected at a volume of 0.5 ml/tube. An aliquot of each fraction was subjected to gel shift assay for p53CP activity.
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The p53CP-containing fractions were combined, dialyzed, concentrated and loaded onto a SDSPAGE gel, followed by Coomassie staining. Four very faint bands with sizes ranging from 40 to 60 kDa (data not shown) were excised and subjected to capillary HPLC-microblotting and Edman sequencing at the Department of Chemistry, Pfizer Global Research & Development. This approach was unsuccessful due to an insufficient amount of protein (data not shown).
Enrichment of p51/p63 during p53CP purification
Failure to obtain the protein sequence of p53CP by microsequencing prompted us to take an alternative approach to identify p53CP. By that time, two additional p53 family members, p73 and p51/p63, had been cloned and characterized (2023,41) and antibodies against these proteins developed and made commercially available. We then sought to determine whether p53CP is p73 or p51/63, using specific antibodies to probe our p53CP fractions saved at each step of purification. The likelihood that p53CP is p73 is basically excluded for the following reasons: (i) there is an apparent size difference between the two proteins, p73 being ~73 kDa (20,24) whereas p53CP is ~40 or 55 kDa, originally determined by southwestern analysis (35); (ii) in the gel shift assay p73 migrates slower than p53 (24) whereas p53CP migrates faster than p53 (35); (iii) western blot analysis using anti-p73 antibody failed to detect any bands in p53CP fractions after each step of purification (data not shown). The possibility exists that p53CP is p51/p63, since the gene has seven splicing variants (22) and some of the variants have a similar size to p53CP (22,23). This possibility was thus examined. The pooled p53CP-containing fractions from each step of p53CP purification, except DNA affinity chromatography (all p53CP-containing samples were used for the failed protein microsequencing), were subjected to western blot analysis using a specific monoclonal antibody (4A4) recognizing all known p63 variants (Santa Cruz). Protein loading was 80 µg for the nuclear extract and 80 µg for the ammonium sulfate precipitation, 20 µg for the heparinagarose chromatography and 5 µg for the Mono S chromatography fractions. As shown in Figure 4A
, the antibody detects hardly any bands in the nuclear extract. A single distinct band with a size of ~55 kDa was, however, detected in the rest of the fractions, which were enriched after each step of p53CP purification and reached a very high level in the Mono S fraction. This result strongly suggests that p53CP and p51/p63 are co-purified and that p53CP could be p51/p63. Based upon its molecular weight of ~55 kDa, p53CP could be either the p51/p63 isoform TAp63
/p51A or
Np63ß (22,42). To distinguish between them, a p63 polyclonal antibody (N-18) specifically recognizing the N-terminus-containing isoforms was used for western blot analysis. As shown in Figure 4B
, a band with a size of 55 kDa was hardly detected in nuclear extract, but was readily detected in the rest of the fractions. In contrast to what was seen with antibody 4A4, an enrichment of the band density was observed after ammonium sulfate precipitation and heparinagarose chromatography, but not after Mono S chromatography, based upon protein loading (see above), suggesting that other isoforms, lacking the N-terminus portion of the molecule, exist, particularly in the Mono S fraction. Thus, it appears that p53CP is a mixture of the TAp63
/p51A and
Np63ß isoforms.

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Fig. 4. Enrichment of p51/p63 during p53CP purification. The p53CP-containing fractions from the nuclear extract (NE, 80 µg), ammonium sulfate precipitation (AS, 80 µg) and heparinagarose (HA, 20 µg) and Mono S chromatography (MS, 5 µg) were subjected to western blot analysis, probed with a p51/p63-specific monoclonal antibody (4A4) recognizing all isoforms (A) and a p51/p63 polyclonal antibody (N-18) recognizing the N-terminus-containing isoforms (B).
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Co-purification of p53CP and p51/p63
Following this lead, we next determined whether p53CP co-purified with p51/p63. We reasoned that if p53CP is p51/p63, we should be able to detect p51/p63 protein only in those fractions that contain p53CP activity. Nuclear extracts were prepared from mouse liver tumor cells (H-Tx) that express very high levels of p53CP (35) and directly subjected to DNA affinity purification. Proteins were eluted by increasing NaCl concentrations. Each fraction was then assayed for p53CP activity by gel shift and some of fractions that contained measurable protein were concentrated and assayed for p51/p63 protein by western blot. As shown in Figure 5A
, p53CP activity was detected in two fractions eluted by salt concentrations of 0.30.4 M, which was consistent with the results obtained in HeLa cells (Figure 3
). These are the same fractions where p51/p63 was detected using antibody 4A4 (Figure 5B
). The amount of protein loaded for each fraction was 20 µg for the 0.2 M fraction, 4 µg for the 0.3 M fraction, 4.5 µg for the 0.4 M fraction, 5 µg for the 0.5 M fraction and 3 µg for the 0.7 M fraction. Other fractions contained non-measurable protein levels and were not used. Taken together, these experiments provide strong evidence that p53CP is p51/p63, the third member of the p53 gene family.

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Fig. 5. Co-purification of p51/p63 and p53CP. Nuclear extract was prepared from mouse liver tumor H-Tx cells and directly subjected to DNA affinity column purification. Fractions (0.5 ml/tube) were collected from elution with an increasing concentration of NaCl as indicated. An aliquot from each fraction was subjected to gel shift assay for p53CP activity and the rest of the samples were concentrated and subjected to western blot analysis with a specific antibody (4A4) for p51/p63. The amount of protein loaded in each fraction was as follows: 0.2 M, 20 µg; 0.3 M, 4 µg; 0.4 M, 4.5 µg; 0.5 M, 5 µg; 0.7 M, 3 µg.
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p51/p63, also called Ket, p40 and p73L, was recently cloned by several independent groups (2123,41,43). The gene is localized on chromosome 3q2729, a region that is altered in several cancers, including carcinomas of the lung, cervix, ovaries and head and neck (22,23,43,44). It was recently found that a heterozygous germline mutation in p63 is the cause of EEC syndrome, an autosomal dominant disorder characterized by ectrodactyly, ectodermal dysplasia and facial clefts (45). Mutation of p63 is also responsible for the split-hand/ split-foot malformation (46).
We have previously hypothesized that p53CP could be either a protein that competes with p53 for sequence-specific binding, thus inactivating p53, or have p53-like functions, binding and transactivating p53 downstream target genes (35). It is now clear that p53CP/p51/p63 has p53-like activity to transactivate p53 downstream target genes (27). Potential competition of p53CP/p51/p63 with p53 has also been demonstrated (22). It was found that two p51/p63 splice variants,
Np63
and
Np63
, which lack the transactivation domain, indeed inhibited p53 transactivation activity in a dominant negative fashion (22). The biological significance of p51/p63p53 interaction/competition is starting to emerge. It was recently found that expression of
Np63
, a p63 truncated isoform lacking the N-terminal transactivation domain, was dramatically decreased in normal keratinocytes and newborn epidermis after UVB irradiation. The epidermis, which overexpresses
Np63
in a transgenic mouse model, is more resistant to UVB-induced apoptosis. Thus,
Np63
appears to act in a dominant negative manner against endogenous p53 to decrease p53-mediated, UVB-induced apoptosis in epidermis (47). Likewise,
Np63 was also found to be highly expressed in nasopharyngeal carcinomas (NPC) (48), a cancer where p53 mutation is a rare event but p53 protein levels are quite high (49,50). It was therefore speculated that
Np63 at a high level in NPC inactivates wild-type p53 either by direct competition, direct proteinprotein interaction or heterotetramer formation (48). Finally, p40/AIS, a p51/p63 truncated form that lacks the transactivation domain, was found to induce neoplastic transformation when overexpressed in Rat 1a cells (42). This will be of particular interest in understanding its mechanism of action. Does p40 compete with p53 and act in a dominant negative manner to inhibit p53 function, thus inducing transformation, or do so by a p53-independent mechanism?
We have observed a size discrepancy of p53CP, shown here as an ~55 kDa protein, compared with ~40 kDa reported previously (35). It appears that this 40 kDa protein detected in a crude nuclear extract by southwestern analysis is likely a non-p53CP protein that non-specifically binds to T3SF oligonucleotide (see Figure 1
, NE) which was eliminated after ammonium sulfate purification (Figure 1
, AS). Our previous southwestern analysis also detected a weakly binding band with a size of ~55 kDa in both human and mouse cells, which could be the true p53CP/p51/p63 band (35). Thus, p53CP appears to be p51/p63, most likely the TAp63
/p51A or
Np63ß isoform (22,42) based upon its molecular weight.
In summary, we have partially purified p53CP and found it as a very low abundance protein in HeLa cells. We have characterized p53CP as p51/p63, which has both p53-like and p53-competing activities. We have determined that p53CP is also equivalent to another nuclear protein, called non-p53 p53RE-binding protein (51), and that they all belong to the p51/p63 group (52). Identification of p53CP/non-p53 p53RE-binding protein as p51/p63 clarifies the confusion in their identity (34,53) and facilitates a thorough study of p53CP function as a potential p53-competing protein.
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Notes
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2 Present address: Abilene Christian University, Box 27868, Abilene, TX 79699, USA 
3 To whom correspondence should be addressed Email: yi.sun{at}pfizer.com 
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Acknowledgments
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We would like to thank Dr Hua Lu at the Oregon Health Science University for communicating results prior to publication.
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Received August 22, 2000;
revised October 11, 2000;
accepted October 16, 2000.