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[Cancer Research 59, 5902-5907, December 1, 1999]
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[Cancer Research 59, 5902-5907, December 1, 1999]
© 1999 American Association for Cancer Research

Advances in Brief

Deletion of the COOH-Terminal Region of p73{alpha} Enhances Both Its Transactivation Function and DNA-binding Activity but Inhibits Induction of Apoptosis in Mammalian Cells1

Toshinori Ozaki, Masahiko Naka, Naoyuki Takada, Mitsuhiro Tada, Shigeru Sakiyama and Akira Nakagawara2

Division of Biochemistry, Chiba Cancer Center Research Institute, Chiba 260-8717 [T. O., M. N., N. T., S. S., A. N.], and Division of Cell Biology, Cancer Institute, Hokkaido University School of Medicine, Sapporo 060-8638 [M. T.], Japan


   ABSTRACT
Top
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
The candidate tumor suppressor p73 has a high sequence homology with p53 within the NH2-terminal transactivation domain, the sequence-specific DNA-binding region, and the oligomerization domain. However, p73{alpha}, which is most abundantly expressed in many tissues and cells among the alternatively spliced forms of p73, has an additional long COOH-terminal tail that might distinguish the function of p53 and p73{alpha} or other p73 splicing variants. To examine the functional role of the p73{alpha} COOH-terminal region, we generated a series of p73{alpha} truncation mutants including p73{alpha}(1–247) (retaining only a transactivation domain), p73{alpha}(1–427) (lacking the most COOH-terminal region including a SAM domain), and p73{alpha}(1–548) (deleting an extreme COOH-terminal region except a SAM domain). When transfected into COS cells, all of p73{alpha}, p73{alpha}(1–548), and p73{alpha}(1–427) localized in the cellular nucleus, whereas p73{alpha}(1–247) localized in both nucleus and cytoplasm. Intriguingly, when compared with p73{alpha}, both p73{alpha}(1–427) and p73{alpha}(1–548) showed a significant stimulation of the transcription of luciferase reporters harboring three p53-responsive promoters (p21Waf1, Mdm2, and Bax) in p53-deficient SAOS-2 cells. Gel retardation assays showed that DNA-binding activity of p73{alpha}(1–427) and p73{alpha}(1–548) was increased as compared with that of the full-length p73{alpha}. However, the colony formation assays using SAOS-2 cells demonstrated that, contrary to p73{alpha}, transfection of p73{alpha}(1–427) or p73{alpha}(1–548) resulted in no significant reduction of the number of colonies. These suggest that the distal COOH-terminal region of p73{alpha} is a cis- or trans-acting regulatory domain and regulates its functions diversely.


   Introduction
Top
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 REFERENCES
 
A novel gene for the first homologue of tumor suppressor p53, termed p73, has been cloned recently, and it has been shown that, similarly to p53, p73 induces both G1 arrest of the cell cycle and apoptosis in mammalian cells. However, the difference between biological roles of p53 and p73 is unclear. One of the structural differences between p53 and p73 is the presence of the additional COOH-terminal region in p73. In contrast to p53, p73 has at least four splicing variants: p73{alpha} (636 amino acids), p73{beta} (499 amino acids without exon 13), p73{gamma} (475 amino acids without exon 11), and p73{delta} (403 amino acids without exons 11–13) (1 , 2) . Although three major domains of p53 (NH2-terminal transactivation domain, DNA-binding domain, and oligomerization domain) are conserved in all four p73 splicing variants, the additional COOH-terminal regions are varied by alternative splicing. p73{alpha} possesses an additional 216-amino acid segment (codons 421–636) at its COOH-terminus, which includes a putative protein-protein interaction motif, SAM domain (codons 484–549; Ref. 3 ), whereas p73{beta} lacks most of the SAM domain by splicing out exon 13. Interestingly, p51/p63 (a human homologue of the rat Ket gene), an additional member of the p53-related genes, also has various splicing variants (4, 5, 6, 7) .

Because p73 shares significant sequence similarity with p53, its function may closely resemble that of p53 (8 , 9) . Ectopic overproduction of p73{alpha} or p73{beta} not only activates the transcription of p21Waf1/Cip1 but also induces apoptosis in cells irrespective of their p53 status (1 , 10) . The structural integrity of the p73 DNA-binding domain is required for these activities, suggesting that p73 recognizes and activates the p53-responsive DNA element. Indeed, p73 is able to transactivate various endogenous targets of p53 to various degrees, such as RGC (ribosomal gene cluster), Mdm2, Bax, cyclin G, GADD45, IGF-BP3 (insulin-like growth factor binding protein 3), and 14-3-3{sigma} (10, 11, 12, 13, 14) . Interestingly, p73 associates with p53 in a yeast-based, two-hybrid assay (1) . Recent data have provided an evidence that naturally occurring p53 mutants but not wild-type p53 are stably associated with p73{alpha} and reduce its transactivation and apoptosis-inducing functions (12) .

On the other hand, a number of remarkable differences between p73 and p53 have been reported. p73 differentially transactivated some but not all p53-responsive genes (11) . The levels of p73 are not changed by the exposure to DNA-damaging agents such as actinomycin D or UV irradiation, which increase the p53 levels (1) . The steady-state levels of p73 are not reduced by the complex formation with Mdm2 (13 , 14) , which induces the ubiquitination and proteolytic degradation of p53 (15, 16, 17) . Furthermore, viral oncoproteins, including adenovirus E1B, SV40 large T antigen, and human papillomavirus E6, do not interact with p73, although these viral proteins bind to wild-type p53 to inhibit its activities (18, 19, 20) . These findings strongly suggest that biological functions of p73 and p53 differ under physiological conditions.

The p73 gene has been mapped to the short arm of chromosome 1 (1p36.33), a region that is frequently deleted in a variety of human cancers (1 , 21 , 22) . However, extensive mutation searches have revealed that mutations are rare in the coding region of p73 (1 , 23, 24, 25, 26, 27, 28, 29, 30) . Nevertheless, we have found two missense mutations (P405R and P425L) in the COOH-terminal region of p73 in 140 primary neuroblastomas (28) . The region (amino acid residues 382–491) contains a glutamine- and proline-rich domain and exhibited a transactivation function in a GAL4 DNA-binding fusion system (31) . Interestingly, this transactivation activity was significantly impaired in those mutant forms of p73, suggesting the presence of functional significance of the COOH-terminal region of p73 (31) .

In the present study, we found that deletions of the p73{alpha} COOH-terminal region enhanced both the transactivation function and the DNA-binding activity. However, standard colony formation assays unexpectedly showed that removal of the COOH-terminal region resulted in a significant reduction of the growth-suppressive activity of p73{alpha}.


   Materials and Methods
Top
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 REFERENCES
 
Cell Culture.
COS, SAOS-2, and 293 cells were cultured at 37°C in DMEM supplemented with 10% heat-inactivated fetal bovine serum and kanamycin.

Plasmids.
The mammalian expression vector encoding HA3 -tagged p73{alpha} or p73{beta} was a gift from Mourad Kaghad (Sanofi Recherche, Paris, France). To generate truncated forms of p73{alpha}, the HindIII-EcoRI, HindIII-HincII, or HindIII-EcoNI restriction fragment from pcDNA3-p73{alpha} was filled in with Klenow enzyme and ligated to the EcoRV site of pcDNA3 to give pcDNA3-p73{alpha}(1–247), pcDNA3-p73{alpha}(1–427), or pcDNA3-p73{alpha}(1–548), respectively.

DNA Transfection.
SAOS-2 cells (5 x 105 cells/dish) were transfected by the calcium-phosphate complex formation protocol with a total of 20 µg of DNA, essentially as described (32) . COS cells (1 x 105 cells/dish) were transfected with a cationic lipid preparation (Fugene 6; Boehringer Mannheim Biochemicals, Indianapolis, IN), according to the manufacturer’s protocol. Two µg of plasmid DNA were used for each transfection.

Subcellular Localization.
Transiently transfected COS cells were fixed with PBS containing 3.7% formaldehyde and then rendered permeable with PBS containing 0.1% Triton X-100 at room temperature for 5 min. Cells were treated with blocking solution (3% BSA in PBS) at room temperature for 1 h to block nonspecific antibody-binding sites. Then the cells were incubated with monoclonal anti-HA (12CA5) antibody (diluted 1:100; Boehringer Mannheim Biochemicals). Antibodies were stained with rhodamine-conjugated goat antimouse secondary antibody (diluted 1:200; Life Technologies, Inc., Grand Island, NY).

Western Blotting Analysis.
Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with monoclonal anti-HA (12CA5) antibody (diluted 1:1000; Boehringer Mannheim Biochemicals) in 5% nonfat milk, 50 mM Tris-Cl (pH 7.6), 100 mM NaCl, and 0.1% Tween 20, followed by incubation with a horseradish peroxidase-conjugated goat antimouse secondary antibody (diluted 1:4000; Jackson ImmunoResearch Laboratories, West Grove, PA).

Reporter Gene Assay.
WWP-luc(p21WAF1-luc; Ref. 33 ), pGL2-NA(Mdm2)-luc (34) , and pGL3-Bax-luc (35) were used as reporter constructs. For transfection, SAOS-2 cells (5 x 105 cells/dish) were transfected with one of the expression vectors (10 µg of each) and two different reporter constructs, including a cytomegalovirus-derived Renilla luciferase cDNA (1 µg) and a p53-responsive firefly luciferase cDNA (10 µg). Forty-eight h after transfection, luciferase activity was determined by a dual-luciferase reporter gene assay system (Promega Corp., Madison, WI), following the manufacturer’s instructions.

In Vitro Translation and Gel Retardation Assay.
In vitro translation was carried out with the TNT translation system (Promega), according to the manufacturer’s procedure. For electrophoretic mobility shift assay, a 27-mer double-strand oligonucleotide containing a p53 DNA-binding site (5'-TACAGAACATGTCTAAGCATGCTGGGG-3'; Santa Cruz Biotechnology, Santa Cruz, CA) was radiolabeled with [{gamma}-32P]ATP by using polynucleotide kinase (Takara, Otsu, Japan). The binding of the translation products to the radiolabeled oligonucleotide was performed in 12.5 mM Tris-Cl (pH 7.5), 50 mM KCl, 3.125 mM MgCl2, 0.25 mM EDTA, 0.5 mM DTT, 5% glycerol, 100 µg/ml of poly(deoxyinosinic-deoxycytidylic acid) (36) . The reaction mixtures were incubated for 15 min on ice and for an additional 15 min at room temperature. The reaction mixtures were resolved on a 5% native polyacrylamide gel in 1x Tris-borate-EDTA buffer at room temperature.

Colony Formation Assay.
SAOS-2 cells (5 x 105 cells/dish) were transfected by the calcium phosphate/DNA precipitation method, as described above. Drug-resistant colonies were selected by growth in G418 (400 µg/ml) for 2 weeks. The G418-resistant colonies were then stained with Giemsa’s solution and counted. The experiments were repeated three times.


   Results
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 REFERENCES
 
Construction of the COOH-Terminal Deletion Mutants of p73{alpha} and Their Subcellular Localizations.
To examine the possible biological significance(s) of the COOH-terminal region of p73{alpha}, we constructed various expression vectors encoding HA-p73{alpha} derivatives that lacked a series of COOH-terminal regions (Fig. 1A)Citation . These expression plasmids were then subjected to the in vitro transcription and translation reactions. As shown in Fig. 1BCitation , p73 derivatives of the expected sizes and comparable amounts were obtained. All of these plasmids were transfected independently into 293 cells, and total cell lysate derived from each transfection was subjected to Western analysis by using a monoclonal anti-HA antibody. Each expression plasmid gave rise to a stable protein with the expected size (Fig. 1C)Citation . To evaluate the subcellular localization of each truncated form of p73, each expression plasmid under the control of cytomegalovirus promoter was transiently transfected into COS cells. Forty-eight h after transfection, cells were fixed and analyzed by the anti-HA immunofluorescence staining. Each exogenous p73 species except p73{alpha}(1–247) appeared to be localized in the cell nucleus (Fig. 2)Citation , whereas p73{alpha}(1–247) was detected both in the cell nucleus and the cytoplasm. Similar results were also obtained in Western analysis using nuclear and cytoplasmic fractions of the transfected cells (data not shown).



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  		Fig. 1. Structure of derivatives of p73 studied and their expression. A, N-terminal transactivation domain (TA; residues 1–54) and site-specific DNA-binding region (DB; residues 131–310) are shown by dotted boxes and gray boxes, respectively. Oligomerization domain (OD; residues 345–380) and SAM domain (residues 484–549) are indicated by hatched boxes. Additional five amino acids specific for p73{beta} are depicted by the filled box. B, expression of truncated forms of p73. In vitro translated products were separated on an SDS-PAGE (10% gel) and subjected to Western analysis with monoclonal anti-HA antibody. Arrowheads, expected products. C, 293 cells were transfected transiently with expression plasmids encoding indicated p73 proteins or with the backbone expression plasmid (pcDNA3). Total cell extracts were resolved by electrophoresis in 7.5% SDS-polyacrylamide gel and immunoblotted with a monoclonal anti-HA antibody. Arrowheads, positions of the expected products.

 


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  		Fig. 2. Subcellular localization of p73 derivatives. COS cells were transfected with 2 µg of each expression plasmid encoding HA-tagged p73{alpha}, p73{beta}, p73{alpha}(1–247), p73{alpha}(1–427), or p73{alpha}(1–548). Forty-eight h after transfection, cells were processed for immunofluorescence using monoclonal anti-HA antibody and rhodamine-conjugated secondary antibody.

 
The COOH-Terminal Deletions of p73{alpha} Enhance p53-responsive Promoters in SAOS-2 Cells.
To determine the effects of the COOH-terminal deletions on p73-mediated transcriptional activation, the expression plasmids encoding wild-type p73({alpha} or {beta}) or p73{alpha} COOH-terminal deletion mutants were transiently transfected into a p53-deficient human osteosarcoma cell line (SAOS-2) along with a reporter construct containing a p53-responsive element derived either from human p21Waf1, Mdm2, or Bax promoter. As a control for specificity in these assays, we used a reporter construct that possessed human p21Waf1 promoter lacking the p53-recognition site. In this control experiment, wild-type p53, p73({alpha} or {beta}), or p73 derivatives could not activate luciferase reporter (data not shown). As described previously (12) , p73 splicing variants ({alpha} and {beta}) transactivated all three promoters to various degrees (Fig. 3)Citation . p73{alpha} and p73{beta} were less efficient in activating transcription from the p21Waf1 promoter than p53, whereas the expression of p73{alpha} or p73{beta} resulted in an increase of Bax promoter activation compared with p53. Similar results were also described in another p53-null cell line (H1299; non-small cell lung carcinoma; Ref. 12 ). In the case of Mdm2 promoter, luciferase activity obtained by the exogenous expression of p73{beta} was higher than those of p53 and p73{alpha}, as reported previously (14) . When p73{alpha}(1–247), which lacked the COOH-terminal region and a part of DNA-binding domain, was expressed in SAOS-2 cells, we could not detect transcriptional activation from all of the promoters that we examined. Interestingly, p21Waf1, Mdm2, and Bax promoters were transactivated by p73{alpha}(1–548) more efficiently than by p73{alpha} or p73{beta}. Further deletion to amino acid residue 428 also displayed an enhanced luciferase activity, albeit to a slightly lesser degree than p73{alpha}(1–548).



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  		Fig. 3. Sequence-specific transcriptional activation by p73 derivatives. SAOS-2 cells were transiently cotransfected with the expression plasmid encoding wild-type p53, p73{alpha}, p73{beta}, p73{alpha}(1–247), p73{alpha}(1–427), or p73{alpha}(1–548) along with the luciferase (luc) reporter constructs including p21Waf1-luc (A), Mdm2-luc (B), or Bax-luc (C). Cells were harvested 48 h after cotransfection and subjected to a luciferase assay. Results are shown as fold induction of luciferase activity relative to control cells transfected with an empty vector. The data represent the average of three independent experiments.

 
The COOH-Terminal Deletions of p73{alpha} Enhance DNA-binding Activity.
On the basis of the observations that the COOH-terminal region exhibited the inhibitory effect on the transactivation function of p73{alpha}, it is conceivable that the COOH-terminal region inhibits the sequence-specific DNA-binding activity of p73{alpha}. To evaluate this possibility, the binding of these truncated forms of p73{alpha} to the p53 recognition site was determined by electrophoretic mobility shift assays. A radiolabeled p53 DNA-binding element was incubated with p73{alpha}, p73{beta}, p73{alpha}(1–247), p73{alpha}(1–427), or p73{alpha}(1–548) generated in vitro by using rabbit reticulocyte lysate. Unprogrammed rabbit reticulocyte lysate was used as a negative control. As shown in Fig. 4ACitation , the DNA-protein complex contained HA-tagged p73{beta}, because it disappeared in the presence of a monoclonal anti-HA antibody. This observation indicated that p73{beta} can interact with the p53 recognition site as reported previously (1) . p73{alpha}(1–247), which lacked the COOH-terminal region, the oligomerization domain, and a part of DNA-binding domain, did not bind to the radiolabeled p53-responsive element (Fig. 4B)Citation . The DNA-binding activity of p73{alpha} was quite weak in our experimental conditions. However, p73{beta} exhibited much higher DNA-binding activity. This suggested that the additional COOH-terminal region that was lost in p73{beta} inhibited the complex formation between p73 and the p53-responsive element. This possibility was further confirmed by the gel retardation assays using p73{alpha}(1–427) or p73{alpha}(1–548) because they showed much higher DNA-binding activity than p73{alpha} (Fig. 4B)Citation .



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  		Fig. 4. Electrophoretic mobility shift assay. A, in vitro-translated HA-tagged p73{beta} was used in a gel mobility shift assay with a radiolabeled, p53-responsive element. The reaction was performed in the absence (Lane 2) or in the presence (Lane 3) of a monoclonal anti-HA antibody. Lane 1, a control reaction using rabbit reticulocyte lysate incubated with backbone plasmid (pcDNA3). Arrowhead, position of p73{beta}-DNA complex. B, HA-tagged p73{alpha} (Lane 2), p73{beta} (Lane 3), p73{alpha}(1–247) (Lane 4), p73{alpha}(1–427) (Lane 5), or p73{alpha}(1–548) (Lane 6) generated by using rabbit reticulocyte lysate was used in a gel mobility shift assay with a radiolabeled p53-responsive element. Arrowheads, positions of p73 complexes.

 
Inactivation of Growth-suppressive Activity of p73{alpha} by the COOH-Terminal Deletions.
A possible role(s) of the COOH-terminal region of p73{alpha} in the growth suppression was examined by the colony formation assays. The expression plasmid encoding p73{alpha}, p73{beta}, or p73{alpha} COOH-terminal deletion mutants was transfected into SAOS-2 cells. pcDNA3 and the chimeric plasmid harboring wild-type p53 cDNA were used as a negative and a positive control, respectively. The transfected cells were selected in the presence of G418. After 2 weeks of selection, the plates were fixed and stained with Giemsa’s solution, and numbers of G418-resistant colonies were scored. As shown in Fig. 5Citation , overexpression of wild-type p53, p73{alpha}, or p73{beta} resulted in a massive reduction in number of colonies, as compared with cells that were transfected with the backbone expression vector. In contrast, many G418-resistant colonies were observed after transfection with the expression plasmid encoding p73{alpha}(1–427) or p73{alpha}(1–548). The number of colonies generated by the exogenous expression of p73{alpha}(1–247) was almost the same as that of the control transfection (pcDNA3).



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  		Fig. 5. Colony formation assay. SAOS-2 cells were transfected with either an empty control vector (a) or expression vector encoding wild-type p53 (b), p73{alpha} (c), p73{beta} (d), p73{alpha}(1–247) (e), p73{alpha}(1–427) (f), or p73{alpha}(1–548) (g). Stably transfected cells were selected with G418 for 2 weeks, and numbers of G418-resistant colonies were counted after staining with Giemsa’s solution. One of three independent experiments is shown (A). The effect of the overexpression of wild-type p53, p73{alpha}, p73{beta}, or p73{alpha} deletion variants was measured by comparing the number of drug-resistant colonies relative to that generated by the transfection with the control pcDNA3 plasmid (B).

 

   Discussion
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
REFERENCES
 
The NH2-terminal region of p73 acts as a transcriptional transactivator (31) , and the disruption of the p73 DNA-binding region results in a marked reduction of the transactivation function (10) . However, p73 has an extended COOH-terminal region that is absent in p53 and is varied among the alternatively spliced variants of p73 thus far known (1) . Homotypic and/or heterotypic interactions between p73 splicing variants has been demonstrated in a yeast-based, two-hybrid assays (1 , 2) . In addition, the naturally occurring missense mutations have been found in the COOH-terminal region of p73 in primary neuroblastomas (28) . In the present study, we examined function of p73 by focusing on the role of its COOH-terminal region and found that p73{alpha} with deletion of codons 428–636 as well as codons 549–636, which lacked the extreme COOH-terminal region with or without the SAM domain, respectively, enhanced both transactivation function and DNA-binding activity in mammalian cells. However, those deletions unexpectedly inhibited suppression of the colony-forming activity. These have suggested that the COOH-terminal tail of p73{alpha} is a regulatory domain that functions as a positive or negative regulator, possibly in a cis- or trans-acting manner.

Our transient transfection experiments revealed that p73{alpha}(1–247) localized in both nucleus and cytoplasm, whereas p73{alpha}(1–427) localized only in the nucleus, suggesting that the nuclear localization signal(s) exists within the region between residues 248 and 427. As described previously, three potential NLSs reside in the COOH-terminal region of p53 (NLS I, II, and III; Ref. 37 ). Among them, NLS I (316-PQPKKKP-322) alone may direct the pyruvate kinase fusion protein to the nucleus (38) . The similar short sequence containing the basic residues (338-PALGAGVKKRR-348) is also present in p73, and it could be one of the potential NLSs.

The present results obtained from the luciferase reporter assays under the control of three p53-responsive promoters (p21, Mdm2, and Bax) in p53-null SAOS-2 cells showed that the COOH-terminal deletions up to residue 428 resulted in a dramatic enhancement of the transactivation ability of p73{alpha}, irrespective of promoters tested. In addition, both p73{alpha}(1–427) and p73{alpha}(1–548) bound to the p53-responsive DNA element much more efficiently than the full-length p73{alpha}, as determined by gel retardation assays. Those results indicated that the regulation of DNA-binding activity of p73 by its COOH-terminal region was well correlated with that of the transactivation function by the same region. As already known, the COOH-terminal region of p53 (residues 311–393) is composed of at least two structural determinants, the oligomerization domain (residues 319–360) and an adjacent basic region (residues 363–393). Three-dimensional data suggest that the COOH-terminal region exists in close proximity to the central DNA-binding domain (39) . Furthermore, various lines of evidence indicate that structural modifications within the COOH-terminal region of p53, which are caused by antibody binding, phosphorylation by cyclin-dependent kinases or casein kinase II, acetylation by p300, glycosylation, or COOH-terminal deletions, markedly stimulate the sequence-specific DNA-binding activity (40, 41, 42, 43, 44) . Zhou et al. (36) have demonstrated that the last 50 amino acids of p53 COOH-terminal region are essential for the transcriptional transactivation. Although the COOH-terminal segment of p73{alpha} showed no significant sequence homology with that of p53, our results suggest that it has an inhibitory role for the sequence-specific transactivation function of p73{alpha}.

p73{alpha} showed a weak DNA-binding activity, maybe attributable to the presence of the inhibitory element described above. On the other hand, p73{beta}, which possesses the much shorter COOH-terminal segment than p73{alpha} by alternative splicing and contains the additional five amino acids at its COOH terminus, exhibited the comparable levels of the DNA-binding activity with those of p73{alpha}(1–427) and p73{alpha}(1–548). However, its transactivation activity was lower than those of the COOH-terminal truncation mutants. These observations might suggest the existence of an alternative regulatory mechanism for the transactivation function of p73{beta}.

In p53, the sequence-specific transactivation function has been shown to be essential for the ability of p53 to suppress cell growth (45, 46, 47) . On the other hand, some investigators have found that the transcriptional activation is not required for p53-induced apoptosis (48 , 49) . Our results demonstrated that the growth-suppressive activity of p73{alpha} was significantly reduced by the COOH-terminal deletions, although these deletions caused the remarkable increases of both the transactivation and the DNA-binding abilities of p73{alpha}. Because p73{alpha}(1–247) with an impaired sequence-specific transactivation function was completely defective in the growth suppression, the transcriptional activation toward p53-responsive promoters is prerequisite for the cell growth repression by p73{alpha}, as supported by the previous reports (1 , 2) . Interestingly, both p73{alpha} and p73{beta} had relatively lower transactivation ability than the p73{alpha} with COOH-terminal deletion mutants, but they exhibited remarkable growth suppression. It is intriguing to note that the expression of proliferating cell nuclear antigen is regulated differentially by the levels of wild-type p53; lower levels of p53 expression activate the proliferating cell nuclear antigen promoter, whereas its higher levels do not (50 , 51) . It is possible that the enhanced transactivation ability of p73{alpha} caused by the COOH-terminal deletions may lead to the induction of some targets that are involved in growth promotion. Thus, the structural integrity of the COOH-terminus might be responsible for the growth suppression by p73{alpha}.

Recently, it has been shown that tumor-derived p53 mutants were able to reduce the apoptosis-inducing activity of p73{alpha} through the direct interaction with p73{alpha} (12) . Additionally, Mdm2 disrupted the association of p73 with p300 and repressed the p73 function (13) . These observations strongly suggest that protein-protein interactions are closely involved in the regulation of p73. Therefore, the identification of protein(s) that can associate with the COOH-terminal region may provide a clue for better understanding of the p73-mediated cellular response.


   FOOTNOTES
 
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.

1 This work was supported in part by a Grant-in-Aid from the Ministry of Health and Welfare for a New 10-Year Strategy for Cancer Control, a Grant-in-Aid for Scientific Research on Priority Areas, and a Grant-in-Aid for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture, Japan. N. T. is an Awardee of the Research Resident Fellowship from the Foundation for Promotion of Cancer Research in Japan. Back

2 To whom requests for reprints should be addressed, at Division of Biochemistry, Chiba Cancer Center Research Institute, 666-2, Nitona, Chuoh-ku, Chiba 260-8717, Japan. Phone: 81-43-264-5431; Fax: 81-43-265-4459; E-mail: akiranak{at}chiba-cc.pref.chiba.jp Back

3 The abbreviations used are: HA, hemagglutinin; NLS, nuclear localization signal. Back

Received 7/28/99. Accepted 10/18/99.


   REFERENCES
Top
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

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