Carcinogenesis vol.22 no.2 pp.295–300, 2001
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
1Department of Biological Chemistry, University of Michigan, Ann Arbor,
MI 48019, USA
2Present
address: Abilene Christian University, Box 27868, Abilene, TX
79699, USA
3To
whom correspondence should be addressed
Email: [email protected]
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
heparin–agarose, 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.
Introduction
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
Abbreviations: DTT, dithiothreitol; NPC, nasopharyngeal carcinomas; PMSF,
phenylmethylsulfonyl fluoride.
© Oxford University Press
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 0–13 bp (6), and transactivates expression of many target genes (7). Growth arrest
induced by p53 is mainly mediated by Waf-1/p21 (8), 14-33σ (9) and PTGF-β (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 (20–23). Like p53, both
p73 and p51/p63 contain regions corresponding to the p53
N-terminal transactivation, central DNA-binding and Cterminal 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
(21,22,24–27). 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 protein–protein
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 (30–33) 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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M.Tan et al.
Materials and methods
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 Tris–HCl, 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.
Heparin–agarose chromatography
Fraction I was applied to an equilibrated heparin–agrose column (25 ml bed
volume) and p53CP was eluted with a linear gradient of KCl (0.1–1.0 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 (0.1–1.0 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 DNA–Sepharose 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 10⫻
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
10⫻ 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 Tris–HCl, 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(dI·dC) 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 (0.05–1 M). Each fraction was monitored by gel shift assay for
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p53CP activity and SDS–PAGE with silver staining for purity determination.
For DNA affinity purification directly from nuclear extract, 5–10 ml of nuclear
extract from a mouse liver tumor line (H-Tx) were dialyzed in cold binding
buffer (20 mM Tris–HCl, 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(dI·dC) was added at
50 µg/ml. The resulting sample was passed through a T3SF–Sepharose 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 (0.1–1 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 SDS–PAGE, followed by Coomassie
staining. Four faint bands with a size range of 40–60 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.
Results and discussion
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 heparin–agrose column.
After sample loading and pre-washing the proteins were eluted
with a linear gradient of KCl (0.1–1 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 48–54. These p53CP-containing fractions were
pooled, dialyzed and loaded onto a Mono S column, eluted
with a linear gradient of KCl (0.1–1.0 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
p53CP is p51/p63
Fig. 1. Purification of p53CP by heparin–agarose chromatography. Nuclear
extract was prepared from HeLa nuclear pellet, precipitated with 40%
ammonium sulfate and loaded onto a heparin–agarose column (bed volume
25 ml). The column was eluted with a linear gradient of KCl (0.1–1 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).
Fig. 2. Purification of p53CP by Mono S ion exchange chromatography.
The p53CP-containing fractions after heparin–agarose purification were
combined, dialyzed and loaded onto a Mono S ion exchange column (bed
volume 1 ml), followed by linear gradient elution (0.1–1 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.
in Figure 2, fractions 43–51, 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(dI·dC). The mixture was
Fig. 3. Purification of p53CP by DNA affinity chromatography. The p53CPcontaining 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 (0.05–1 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.
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 0.05–1 M). Again, an aliquot from each
fraction was used for gel shift assay for p53CP and for SDS–
PAGE with silver staining for purity. As shown in Figure 3,
p53CP was detected in fractions 6–9 (mainly in fraction 8),
corresponding to KCl concentrations of 0.3–0.4 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.
The p53CP-containing fractions were combined, dialyzed,
concentrated and loaded onto a SDS–PAGE 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
(20–23,41) 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.
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M.Tan et al.
Fig. 4. Enrichment of p51/p63 during p53CP purification. The p53CPcontaining fractions from the nuclear extract (NE, 80 µg), ammonium
sulfate precipitation (AS, 80 µg) and heparin–agarose (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).
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 p53CPcontaining 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 heparin–agarose 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 TAp63γ/p51A
or ∆Np63β (22,42). To distinguish between them, a p63
polyclonal antibody (N-18) specifically recognizing the Nterminus-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 heparin–
298
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.
agarose 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 TAp63γ/p51A and ∆Np63β isoforms.
Co-purification of p53CP and p51/p63
Following this lead, we next determined whether p53CP copurified 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 0.3–0.4 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
p53CP is p51/p63
(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 nonmeasurable 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.
p51/p63, also called Ket, p40 and p73L, was recently cloned
by several independent groups (21–23,41,43). The gene is
localized on chromosome 3q27–29, 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, ∆Np63α and
∆Np63γ, which lack the transactivation domain, indeed
inhibited p53 transactivation activity in a dominant negative
fashion (22). The biological significance of p51/p63–p53
interaction/competition is starting to emerge. It was recently
found that expression of ∆Np63α, 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
∆Np63α in a transgenic mouse model, is more resistant to
UVB-induced apoptosis. Thus, ∆Np63α 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 protein–protein 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 TAp63γ/p51A or
∆Np63β 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 p53REbinding 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.
Acknowledgement
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