NBP is the p53 homolog p63
Xiaoya Zeng,
Yong Zhu and
Hua Lu,
Department of Biochemistry and Molecular Biology, Oregon Health Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA
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Abstract
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We previously identified a non-p53, p53-responsive DNA element (p53RE)-binding protein named NBP, functionally analogous to p53, from human cervical carcinoma Hela cells. Here we report a biochemical study demonstrating that this activity is the recently cloned p53 analog p63. NBP was purified through conventional and DNA affinity chromatography to apparent homogeneity with a prominent polypeptide migrating in between the 43 and 68 kDa positions on a SDS gel. This polypeptide immunoreacted with monoclonal anti-p63 but not anti-p53 or anti-p73 antibodies. Also, NBP co-purified with p63 through each step of fractionation, as detected with anti-p63 antibodies. DNAprotein complexes formed with purified NBP and p53RE-containing oligomers derived from the p21waf1 promoter were supershifted by anti-p63 but not anti-p53 antibodies. Thus, these results demonstrate that NBP is encoded by the p53 homolog p63 gene.
Abbreviations: EMSA, electrophoresis mobility shift assay; NBP, non-p53 p53RE-binding protein; p53CP, p53 competing protein; p53RE, p53-responsive DNA element.
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Introduction
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p53, the most frequently altered tumor suppressor protein in human tumors (1), has recently been demonstrated to be a member of a larger family of proteins grouped by sequence similarity and biochemical or cellular functions (24). The other members include p73 and p51/p63 (p63 and p51 are the same; for simplicity, p63 will be used in this paper) (24). At the sequence level two highly conserved regions among these family members are the N-terminal transactivation domain (2229% identity with p53) and the central sequence-specific DNA-binding domain (>68% identity with p53) (24). Functionally, like p53, these homologs bind to the same p53-responsive DNA element (p53RE), which contains two copies of the consensus sequence 5'-RRRCT/AA/TGYYY-3' (5), and exhibit the same transcriptional activity, which is dependent upon this DNA element (4,6) and interaction with a group of transcriptional co-activators CBP/p300 (712). Also, like p53 (1315), these proteins when overexpressed in cells induce apoptosis and growth arrest (4,6). In fact, p73 was shown to induce the same subset of genes as does p53, with a minor difference (6,11,16). Thus, transcriptional and apoptotic functions are well conserved among the p53 family members.
However, unlike p53, p63 and p73 play different physiological roles (1719) and their possible involvement in tumorigenesis is still elusive. p63 is essential for animal development, particularly ectodermal differentiation during embryogenesis (17,18), while p53 knock-out mice develop normally with an early onset of tumorigenesis (20). In contrast, p73-deficient mice display neurological, pheromonal and inflammatory defects without spontaneous tumors (19), suggesting that p73 plays a role in certain specific tissues or physiological processes.
These studies suggest that each of the p53 family members, despite their similar biochemical and cellular functions, must be differentially regulated through separate mechanisms. For example, although MDM2 inhibits the functions of both p53 and p73, it mediates degradation of p53, but not p73, through the ubiquitin-dependent proteosome pathway (11,2126). Conversely, MDM2 was found to enhance p63-dependent transcription (unpublished data). Also, p53 and p73 are regulated by different upstream pathways. For instance, upon
-irradiation p73 is activated by c-Abl tyrosine kinase (2729), while p53 is activated through the ataxia telangiectasia mutatedChk2 pathway (3034). In addition, unlike p53, both p73 and p63 contain a unique C-terminal SAM-like domain which may be involved in proteinprotein interactions (35,36). Although p63 regulation remains to be investigated, distinct mechanisms for regulation of the p53 family members may at least partially account for their different physiological roles in proliferation, differentiation, development, homeostasis and tumorigenesis. Thus, unraveling their regulatory mechanisms is crucial for a better understanding of their biological roles.
We recently identified a p53-like transcriptional factor termed NBP (non-p53 p53RE-binding protein) (37). Similarly, another activity named p53CP (p53 competing protein) was described by others, possessing DNA-binding activity specific for the p53RE sequence (38). NBP is not p73 (37). However, whether NBP is p63 or p53CP remains unclear. In the study described here we have purified this protein to apparent homogeneity through conventional and DNA affinity columns to determine the identity of NBP. Silver staining analysis of the purified NBP revealed a prominent band migrating in between the 43 and 68 kDa molecular weight markers on a denatured SDS gel. Immunoblotting analysis of this protein with antibodies against p53, p73, p63 and N-terminally truncated p40 (a p63 alternative splice form), respectively, showed that this protein only immunoreacted with anti-p63 antibodies. Also, the DNANBP complex specific for the p53RE sequence derived from the p21waf1 promoter (39) was supershifted by an anti-p63, but not anti-p53, antibody. Thus, these results indicate that NBP is the p53 homolog p63.
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Materials and methods
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Cell culture
Human cervical carcinoma HeLa cells were cultured as previously described (40).
Antibodies and reagents
The anti-p40 antibody specifically against the unique N-terminal sequence (N-MLYLENNAQTQFS) of human p40 was provided by David Sidransky (Johns Hopkins Medical School, Baltimore, MD) (41). The monoclonal anti-p63 antibody raised against the N-terminal region (amino acids 1205) of
Np63, which recognizes all the alternative species of p63, was a gift from Frank McKeon (Harvard Medical School, Boston, MA) (4). The polyclonal anti-p63 antibody (N-18, sc8396) specifically against the N-terminal transactivational domain (amino acids 150) of p63, which recognizes p63
, p63ß and p63
but not the N-terminal spliced
Np63s, was purchased from Santa Cruz Biotechnology. Monoclonal anti-p53 antibody Pab421 was described previously (11). Polyclonal anti-p73 antibodies were raised specifically against the C-terminus (amino acids 401636) of p73
and affinity purified as described (42). This anti-p73 antibody recognizes both p73
and p73ß.
Preparation of nuclear extracts
Nuclear extracts were prepared from HeLa cells, using a previously described method (43).
Purification of the p53-like transcriptional factor NBP
Aliquots of 2.4 g nuclear extract (in 140 ml) from ~9x1010 HeLa cells (60l) were used as starting material and fractionated through phosphocellulose P11 and DEAESepharose columns as described previously (37). The p53RE-binding and transcriptional activities of the fractions were assayed by electrophoresis gel mobility shift assay (EMSA) and in vitro transcription reactions as described (40,44). p53RE DNA-binding and transcription activities were detected in both the 0.3 and 0.5 M washes of the first column and the flow-through and 0.3 M fraction from the second column (Figure 1A
). The active fraction (15 mg) was further fractionated on a Mono S (HR10/10; Pharmacia) column which was equilibrated with buffer C 100 (BC100) containing 20 mM TrisHCl, pH 7.9, 0.1 mM EDTA, 10% glycerol, 100 mM KCl, 4 mM MgCl2, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol and 0.25 µg/ml pepstatin A. Proteins were eluted with a linear gradient of KCl from 0.1 to 0.8 M. p53RE-binding activity was detected in several fractions away from the protein peak. The active fractions with less protein were pooled (1.1 mg) and run through a DNA affinity column (0.3 ml bead volume) conjugated with multiple p53 binding sites derived from the p21waf1 promoter (39). Proteins were eluted from this column with BC1000 buffer containing 1000 mM KCl. The active protein fractions were dialyzed against BC50 (50 mM KCl) and stored at 80°C for further analyses.

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Fig. 1. Purification of NBP from HeLa nuclear extracts. (A) Purification procedure for NBP protein from Hela cell nuclear extracts. (B) Silver staining (SS) and western blot (WB) analyses of the fractions from the Mono S and p53RE-specific DNA affinity (W2) columns. Aliquots of 20 µl of active fractions were loaded directly onto a 10% SDS gel, followed by silver staining (left) or western blotting (right) using polyclonal antibodies against p63. MW denotes molecular weight markers as indicated on the left (this is true for all the following figures). (C) EMSA of the active fractions from Mono S FPLC and W2 columns.
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Preparation of p53RE-specific DNA affinity beads
DNA affinity beads covalently coupled with multiple copies of the p53-RE derived from the p21waf1 promoter were prepared based upon a published protocol (45). CNBr-activated Sepharose CL-2B resins (Pharmacia) were used.
EMSA
This assay was performed based on a published method (37,44). The protein components as indicated in the figure legends were incubated with 3'-end-labeled DNA fragments harboring two copies of the p53RE sequence derived from the p21waf1 promoter (5000 c.p.m., 1.0 ng DNA/assay) for 30 min at room temperature. The reaction mixture (20 µl) contained 10 mM HEPES buffer, pH 7.5, 4 mM MgCl2, 60 mM NaCl, 0.1 µg poly(dIdC), 0.1% NP-40 and 0.1 mM EDTA. The complexes formed were separated by electrophoresis on a 4% native gel.
Western blotting
The protein fractions from different columns and nuclear extracts were subject to SDSPAGE. Western blotting was carried out as previously described (37) using antibodies as indicated in the figure legends and proteins were detected with ECL reagents (Amersham).
Silver staining analysis
Protein fractions (20 µl/lane) from the p53RE-specific DNA affinity column were loaded directly onto a 10% SDS gel. Silver staining was conducted as described previously (37).
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Results and discussion
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Purification of NBP from Hela cells
Our previous study suggests that in addition to p73 there exists another p53-like transcriptional factor named NBP, which was partially purified from HeLa nuclear extracts (37). In order to determine the nature of this activity and to characterize it, we further purified this factor by chromatography, monitoring the p53RE-binding activity using EMSA. As shown in Figure 1
, this p53RE-binding activity has been purified to apparent homogeneity through four columns: phosphocellulose P11, DEAESepharose 52, Mono S and p53RE-specific DNA affinity columns. Starting with 2.4 g Hela nuclear extract we ended with 6.0 µg protein containing the p53RE-binding activity. A representative result from the DNA affinity column is shown in Figure 1B
. Silver staining analysis of the active fractions from the Mono S (input of the p21waf1 promoter DNA affinity column) and DNA affinity columns revealed a major polypeptide migrating slower than the 43 kDa marker. This is consistent with our previous result (37), which indicates that NBP activity co-purifies with a protein of ~50 kDa. This polypeptide was neither p53 nor p73, as it did not immunoreact with either anti-p53 or anti-p73 antibodies (37; data not shown). Since another p53 analog, p63 (p51), as well as its alternative splice form p40 without the N-terminal domain, was also recently cloned (3,4,41), we tested whether NBP is p63 or p40. Surprisingly, this 50 kDa polypeptide was detected by both polyclonal and monoclonal anti-p63 antibodies (Figure 1B
, lanes 2 and 3), but not by an antibody specifically against the unique N-terminus of p40 (data not shown). This protein was active in binding to p53RE-containing oligomers to form a DNAprotein complex (Figure 1C
) as well as in a transcription reaction in vitro (37; data not shown). Hence, these results indicate that NBP is the transcriptionally active p53 homolog p63 and not the p40 alternative.
Co-purification of NBP with p63 but not p53 and p73
To ensure that it is p63 and not p53 or p73 that co-purifies with NBP activity, we next reprobed the fractions from each step of the purification with antibodies raised against p53, p63 and p73. As shown in Figure 2A
, both p53 and p73 were detectable in the nuclear extracts and fractions from the first column (lanes 2 and 3), but not in the DEAESepharose 52 and Mono S columns (lanes 4 and 5), indicating that p63 was separated from p53 and p73 on the DEAESepharose 52 column. In contrast, p63 was detected throughout each step of purification, co-purifying with the p53RE-binding activity (Figure 2B
). During fractionation we detected several different p53REprotein complexes in fractions from the phosphocellulose column (data not shown and Figure 2B
, lane 5) as well as in DEAESepharose 52 fractions (Figure 2B
, lane 6). Because we primarily traced complexes migrating faster than the p53DNA complex as indicated in Figure 2B
, we were able to purify the NBP activity (Figure 1
). For the purpose of achieving better purification, we specifically selected from the Mono S column those fractions that contained fewer proteins for further fractionation on the p53RE-containing DNA affinity column. This is why the input Mono S fractions for the affinity column (Figure 1C
, lane 2) did not display the non-specific complex detected in the Mono S fractions used in the experiments in Figure 2B
(lane 7). This strategy was helpful in purifying NBP (Figure 1B
, left, lane 3). Taken together, these results show that the NBP activity co-fractionates with the p53 homolog p63, thus termed p63NBP.

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Fig. 2. Co-purification of NBP with p63. (A) Western blot analysis of the fractions from each step of the purification. Aliquots of 25 µl of each fraction were loaded directly onto a 10% SDS gel, followed by western blotting using antibodies against p73, p53 (Pab421) or p63. S100 denotes cytoplasmic extracts; NE is the nuclear extracts; P11, DE and MS represent the phosphocellulose, DEAESepharose and Mono S columns, respectively. (B) EMSA analysis of the active fractions from each step of purification. An aliquot of 2 µl of each protein sample was used in the DNA-binding assay as described in Materials and Methods. #, a non-specific complex. An aliquot of 50 ng p53 purified from baculovirus-infected SF9 cells was used as a control in lane 2.
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Effect of anti-p63 antibodies on the p53RE-binding activity of NBP
To test whether a DNAprotein complex is indeed formed with p63 protein and is specific for the p53RE sequence, we next conducted an EMSA experiment with non-labeled p53RE-containing oligomers as competitors and using antibodies against p63 or p53. As shown in Figure 3
, the oligomers containing wild-type but not mutant p53RE sequence derived from the p21 promoter specifically inhibited formation of the p53RENBP complex (lanes 35). The GADD45 promoter-derived p53RE oligomers, however, had less effect on this complex (lane 6), consistent with previous results (37). This complex was supershifted and enhanced to some degree by anti-p63, but not anti-p53, antibody (lanes 7 and 8). Noticeably, the p63NBPp53RE complex migrated faster than did the p53p53RE complex (compare lane 2 with lane 3), indicating that the former complex is smaller than the latter. From these results we can conclude that the NBPDNA complex contains p63 and is specific for the p53RE binding sequence.

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Fig. 3. The NBPp53RE complex is supershifted by anti-p63 but not anti-p53 antibody. Aliquots of 2 µl of NBP purified through a p53RE DNA affinity column were used in this EMSA experiment. An aliquot of 50 ng p53 was used in lane 2. Poly(dIdC) (0.5 µg) was used as non-specific competitor in all the reactions and, additionally, 0.2 µg specific DNA competitors as indicated at the top were used in lanes 46. Aliquots of 0.5 µg polyclonal anti-p63 or Pab421 antibodies as indicated at the top were used.
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The results from this study demonstrate that our previously identified NBP activity is the p53 homolog p63. At least three lines of evidence support this conclusion. First, the purified NBP was primarily composed of a singular polypeptide with a molecular weight of ~50 kDa, as revealed by silver staining (Figure 1B
). This polypeptide immunoreacted with anti-p63, but not anti-p53 or anti-p73, antibodies (Figure 1B
). Second, reprobing the active fractions from each step of the purification with these antibodies confirmed that NBP co-purified with p63, but not p53 or p73 (Figure 2A
). Finally, formation of a p53RENBP complex was specifically supershifted by the anti-p63 antibody but not by the anti-p53 antibody Pab421 (Figure 3
). Since the polyclonal anti-p63 antibody used in this study was specific for the N-terminus of this protein and the anti-p40 antibody did not react with NBP (data not shown), the NBP protein was not the splice alternative p40, which lacks the N-terminal transactivation domain (41) and inhibits p53-, p63- and p73-dependent transcription (4). There are several alternatively spliced forms of p63, such as p63
, p63ß and p63
(3,4). Because p63
and p63ß are much larger than p63
, NBP is probably the p63
form. Alternatively, NBP may be a shorter splice form of the p63 family members. Recently, another p53-like protein, p53CP, originally identified from murine cells (38), was partially purified by another group and also found to immunoreact with anti-p63 antibodies (46). Therefore, NBP and p53CP appear to be the same activity encoded by the p53 analog p63.
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Notes
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1 To whom correspondence should be addressed Email: luh{at}ohsu.edu 
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Acknowledgments
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We thank Kathleen M.Alexander for her secretarial expertise, David Sidransky and Frank McKeon for generously providing anti-p40 and anti-p63 antibodies and Yi Sun for communicating the p53CP study prior to publication. This work was supported by grants to H.L. from the NIH and ACS.
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Received August 18, 2000;
revised October 3, 2000;
accepted October 10, 2000.