J. Mol. Biol. (1996) 260, 623–637

REVIEW ARTICLE

Structural Aspects of the p53 Protein in Relation to Gene Evolution: A Second Look Thierry Soussi1* and Pierre May2 1

Unite´ 301 INSERM and Universite´ P. & M. Curie Institut de Ge´ne´tique Mole´culaire, 27, rue J. Dodu 75010 Paris, France 2 UMR 217, Laboratoire de Cance´roge´ne`se Mole´culaire DRR, SDV, CEA, 92265 Fontenay aux Roses, Cedex France

*Corresponding author

Several years ago, a comparison of the amino acid sequences of p53 proteins from a variety of species enabled us to reveal structural features of this protein, giving clues to its function. Since then, numerous studies on the biochemical, immunological and biological functions of p53 as well as on its structure (including crystallography data) have provided considerable insight into the multifunctional aspects of p53. The purpose of this review is to present the most recent data concerning the various structural features of the p53 protein with special emphasis on its flexibility, which plays a key role in regulation of its biological activity. 7 1996 Academic Press Limited

Keywords: p53 tumor suppressor gene; transcription factor; conformation flexibility; DNA damage recognition; cell cycle checkpoint

Introduction Born in 1979 of an encounter between the virology of DNA tumor viruses and the immunology of transplantation antigens, p53 spent a rather lonely childhood as a tumor antigen overexpressed in various transformed or tumor cell lines (Crawford, 1983). After a brief sojourn amidst the oncogenes, it finally found its legitimate place within the family of tumor suppressor genes, while still maintaining a foothold in the oncogene world (Jenkins & Stu¨rzbecher, 1988; Lane & Benchimol, 1990). Yet unlike the Rb gene, which is the straight archetype of tumor suppressor genes, the p53 gene has some original features. More than 95% of the alterations in the p53 gene are point mutations that produce a mutant protein, which in most cases has lost its transactivational activity (Crook et al., 1994; Ory et al., 1994). Nevertheless, the synthesis of the mutant p53 protein is not always harmless for the cell. However, it has been shown that some p53 mutants (depending on the site of mutation) exhibit a dominant negative phenotype and are able to associate with the wild-type p53 (expressed by the remaining wild-type allele) to induce the formation of an inactive heterooligomer (the functional form of p53 is a tetramer). Moreover, cotransfection of mutant p53 with an activated ras gene shows that some p53 mutants have high, dominant oncogenic activity, which initially led to the classification of the p53 gene as an oncogene. Without defining any new categories, it is clear that the mechanisms of inactivation of p53 are distinguishable from the 0022–2836/96/300623–15 $18.00/0

classical scheme of inactivation of other tumor suppressor genes. Since 1991, a coherent model has existed to explain the role of p53 in the cellular response to DNA damage (Kastan et al., 1991). In response to this type of damage, the cell induces stabilization of wild-type p53, which in turn can induce the transcription of genes involved in the negative regulation of the cell-cycle and arrest of cell division in the G1 phase (Kastan et al., 1991). After the DNA is repaired the cell-cycle can resume, thereby allowing the replication of damage-free DNA molecules. For reasons that are still unclear, some types of cells are instead induced into a process of apoptosis by this accumulation of wild-type p53. Nevertheless, these different responses all contribute to safeguarding the genetic integrity of the cellular genome. Any cell expressing mutant p53 or not expressing p53 at all is incapable of inducing this genome-stabilizing response. Several target genes of wild-type p53 have recently been identified: the WAF1-CIP1 gene, which codes for a 21 kDa protein that specifically inhibits the kinase activity of the cdk2-cyclin complex required for the G1 to S transition of the cell-cycle (El-Deiry et al., 1993); the IGF-BP3 gene, which is also a negative regulator of cell proliferation (Buckbinder et al., 1995); the GADD45 gene, which codes for a protein that binds to PCNA (Smith et al., 1994) but with a controversial role in DNA repair (Kazantsev & Sancar, 1995; Kearsey et al., 1995); the Bax gene (Miyashita & Reed, 1995), whose product is involved in the regulation of 7 1996 Academic Press Limited

624 apoptosis; and the mdm2 gene, which regulates negatively p53 protein function (Wu et al., 1993). As we will see below, one of the fundamental aspects of p53 activity is governed by conformational changes in the protein. This has prompted numerous laboratories to perform an extensive series of structure-function studies, one theme of which has been the anatomical dissection of p53 protein, begun in 1983 with the first cloning of human and mouse p53, continued in an exhaustive phylogenetic study leading to the identification of five domains highly conserved during evolution (Soussi et al., 1990) and culminating in the X-ray crystallographic study of human p53 recently performed by Cho et al. (1994). A second theme of these studies deals with conformational changes in p53 in various cellular contexts (Milner, 1994). Thus, the objectives of this review are (1) to update detailed sequence comparison of p53 of different species and the structure-function relationships of p53 in light of recent advances in our knowledge of this protein, and (2) to discuss the implications of these stimulating data as concerns the properties of the mutant p53 proteins found in human cancers (for a more general review, the reader can refer to several recent articles: Greenblatt et al., 1994; Soussi et al., 1994; Cox & Lane, 1995; Elledge & Lee, 1995; Milner, 1995).

Further data on the molecular evolution of p53 Since our first review in 1990 (Soussi et al., 1990), a large number of p53 genes have been characterized. Starting with vertebrates, 23 new p53 genes have been isolated and sequenced, providing a basis for developing new animal models to study this gene. In fact, in the case of the cat, chicken and dog, mutations in the p53 gene have been detected in the central region where mutational hot-spots for human cancer are located. Even more interesting is the identification of the p53 gene in invertebrates such as the squid (Ishioka et al., 1995). Attempts to clone the p53 gene in species as diverse as the sea urchin, Drosophila and yeast have been thus far unsuccessful. By using degenerate oligonucleotides and low-stringency PCR amplification, P. Winge and S. Friend (personal communication) succeeded in cloning p53 in certain molluscs. This result suggests that p53 is found lower in the animal kingdom than previously thought. In view of the importance of this gene, this finding is not unexpected and further p53 genes could be isolated in the near future. It should be noted that although p53 has not been found in yeast, the overexpression of human wild-type p53 inhibits cell division in Saccharomyces cerevisiae and Saccharomyces pombe, whereas mutant p53 does not induce a detectable phenotype (Wagner et al., 1991; Bischoff et al., 1992; Nigro et al., 1992).

Review Article: p53 Protein

Molecular organization of the p53 gene To date, five genes have been cloned in their entirety (Bienz et al., 1984; Lamb & Crawford, 1986; Soussi et al., 1990; Hulla & Schneider, 1993; Albor et al., 1994: Table 1). Their organization is highly similar and they share the following characteristics: (1) The presence of a very large intron (10, 6.1, 6.2, 6.5 and 7.7 kb for man, mouse, rat, hamster and Xenopus) at the 5' end of the gene. For mammalian p53, this intron is located between exons 1 and 2, while in Xenopus, it is found between exons 2 and 3. Its biological significance is totally unknown. (2) Exon 1 is always non-coding. Recently, it has been demonstrated that this region could form a stable stem-loop structure that binds tightly to wild-type p53 but not to mutant p53. This binding inhibits specifically p53 mRNA translation and could provide a means for control of the p53 protein level in the cell (Mosner et al., 1995). (3) The intron/exon distribution between these five genes is globally similar. The introns vary in size but divide up the gene in a very similar manner, with the exception of intron 6, whose counterpart is absent in the rat, and intron 3 in Xenopus, which divides up exons 3 and 4 differently. A pseudogene has been characterized and sequenced in the mouse and corresponds to a copy of mRNA that has been integrated into the cellular genome after reverse transcription (Zakut-Houri et al., 1983). Two similar pseudogenes have been identified in the rat (Hulla, 1992). Alternatively spliced mRNA has been detected in transformed and normal murine cells (Arai et al., 1986; Fukuda & Ogawa, 1992; Kuleszmartin et al., 1994) or in normal human cell (Flaman et al., 1996). It has been demonstrated that the presence of intron 4 within the p53 DNA results in a high level of p53 RNA and protein (Hinds et al., 1989). Similarly, in transgenic mice, p53 gene constructs with intron 4 expressed greater than 100-fold p53 mRNA than constructs without introns (Lozano & Levine, 1991). Intron 4 from the murine p53 gene was shown to be the target of a nuclear protein but its biological significance remains to be determined (Beenken et al., 1991). p53 proteins: phylogenetic comparison and structural implication As early as 1987, the characterization of p53 from Xenopus laevis revealed a certain number of features common to all p53 proteins (Soussi et al., 1987). The subsequent identification of new p53 proteins remarkably confirmed these observations (see Table 1 for references). The p53 protein could be divided into three regions: (1) the amino-terminal region, which contains a large number of acidic residues, no basic residues and a large number of proline residues (including many Pro-Pro pairs); (2) the carboxy-terminal region, which is very hydrophilic and contains many charged residues; (3) the central region of the protein which contains three highly hydrophobic regions and very few

c

Mouse p53 whole cDNA Mouse p53 coding sequence Xenopus p53 domains IV and V PCR amplification PCR amplification PCR amplification

Monoclonal antibody

Mouse p53 whole cDNA

Human p53 whole cDNA PCR amplification PCR amplification PCR amplification PCR amplification NA NA NA PCR amplification PCR amplification Mouse p53 coding sequence Mouse p53 coding sequence

Mouse p53 whole cDNA

Probe

whole cDNA gene and whole cDNA whole cDNA partial cDNA partial cDNA? partial cDNA?

gene and whole cDNA

gene and whole cDNA

whole cDNA whole cDNA partial gene (exon 3 to 8) whole cDNA partial cDNA whole cDNA whole cDNA partial gene (exon 5 and 9) partial gene (exon 4 to 10) partial gene (exon 4 to 10) whole cDNA gene and whole cDNA

gene and whole cDNA

p53 clone available

1.8 kb 2.2 and 3 kb 2.4 kb NA NA NA

2.0 kb

2.0

NAe NA NA NA NA NA NA NA NA NA NA NA

2.8 kb

mRNA size

NA 11 exons NA NA NA NA

10 exons (fusion of exon 6 and 7) 11 exons

NA NA NA NA NA NA NA NA NA NA NA 11 exons

11 exons

Gene structure

367 aa 363 aa 396 aa NA NA 564 aa

387

391

NA NA NA 391 aa 396 aa

393 aa 393 aa NA 386 aa NA

393 aa

p53 protein X02469 (E) X54156 (E) X16384 (E) L20442 (G) S77819 (G) 26608 (G) NA X81705 (E) X81704 (E) U37120 (G) U43902 (G) U44835 (G) NA M75144 (G) U08134 (G) X13058 (E) L07903-910 (G) X00741 (E) X008875-885 (E) X13057 (E) M36962 (G) M75145 (G) U34751 (G) U45237 (G) U43596 (G)

Accession numberb

The first murine p53 cDNAs (Bienz et al., 1984; Jenkins et al., 1984; Pennica et al., 1984; Arai et al., 1986) were shown to contain activating mutations. Finlay et al. (1988) compared all these cDNA with wild-type murine cDNA cloned from non-transformed cells. a The symbol in parentheses has been used in the alignement of Figure 2. b E and G correspond to the accession number for EMBL and GENBANK. respectively. c Matlashewski et al. (1984); Harlow et al. (1985). d Rigaudy & Eckhart (1989). e No information available. f Kay et al. (1994). g Kraegel et al. (1995). h Okuda et al. (1994). i Y. Le Rhun, F. Crechet, M. Martin and E. May, personal communication. j Rivkina et al. (1994). k F. Le Goas, P. Ronco, P. May and C. Caron de Fromentel, personal communication. l Legros et al. (1992). m Soussi et al. (1988b). n Finlay et al. (1988). o Soussi et al. (1988a). p Soussi et al. (1987). q Caron de Fomentel et al. (1992). r P. Winge and S. Friend, personal communication.

Gallus domesticus (CHK)o Xenopus laevis (XL)p Salmo irideus (RT)q Xiphophorus maculatus (XM) Mya arenaria (MY) Loligo forbesi (SQ)p53r

Mus musculus (M)n

Rattus norvegicus (RAT)m

Cercopithecus aethiops (AFG)d Macaca muletta (RH)f Canis familiaris (DG)g Felis catus (FC)h Sus scrofa (PG)i Ovis aries (OV) Bos taurus (BV) Equus caballus (HS) Spermophilus beecheyi (GS)j Marmota monax (WC)j Oryctolagus cuniculus (RB)k Mesocritus auratus (HAM)l

Homo sapiens (H)

Organisma

Table 1. Characteristics of p53 from various species

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Figure 1. Diagram of the various functional domains of human p53. Identified or potential phosphorylation sites at the two extremities of the p53 protein are indicated (Meek, 1994: see the text for more details). In the amino-terminal region, the shaded region corresponds to the mdm2 binding site defined using synthetic peptides (Picksley et al., 1994), while stars correspond to sites of mutations that affect this interaction (Lin et al., 1994). Filled boxes correspond to mutations that affect transactivation, open boxes interaction with the E1b protein (Lin et al., 1994). In the carboxy-terminal region, shaded regions correspond to the three nuclear localization signals (Shaulsky et al., 1990). The two domains required for multimerization of p53 are indicated (Wang et al., 1993; Clore et al., 1994). In the central region, the various amino acid residues that interact directly with DNA are shown (Cho et al., 1994). The DNA sequence is the consensus motif identified by El-Deiry et al. (1992), which was cocrystallized with p53. Filled and open arrows correspond to interaction with DNA backbone and bases, respectively.

charged amino acid residues (Figure 1). Sequence analysis of various p53 proteins shows no signature characteristic of a particular function. On the other hand, comparison of the various proteins shows that their homology is not uniformly distributed, leading to the identification of five blocks highly conserved during evolution. These conserved blocks, defined in 1987 by the comparison of p53 from X. laevis with human p53, have been found in all the p53 proteins characterized to date (Soussi et al., 1987, 1990). Four of these five blocks (II to V) are found in the central region of the protein, while block I is located in the amino-terminal region (Figure 2). These data as a whole suggested that the three regions of the p53 protein might have clearly separate functional roles, with crucial importance of the central region containing blocks II to V (Figure 2). In addition, the amino and carboxy-terminal regions were believed to be located at the surface of the protein, while the central region was stacked within the interior of the molecule. Since that time there has been tremendous progress in

defining the structural and functional features of p53. However, the knowledge and identification of the highly conserved regions remain of central importance in this field (Figure 3). Amino-terminal region of p53 This acidic, proline-rich region contains the transcriptional transactivation domain of the protein (Fields & Jang, 1990; Raycroft et al., 1990). Deletion of amino acid residues 20 to 42 completely abolishes this activity, which can be restored by substituting the transactivation domain of the VP16 protein at the same location (Pietenpol et al., 1994). Functional analysis of this region using a library of point mutations at various residues has shown, surprisingly, that a single point mutation is generally not able to completely abolish transactivational activity (Lin et al., 1994). On the other hand, double or triple mutants are totally inactive. Residues Leu22 and Trp23 are crucial for this transactivational activity (Lin et al., 1994). These

Review Article: p53 Protein

627

Figure 2. Colocalization between highly conserved domains (Soussi et al., 1990), structurally essential regions of p53 (Cho et al., 1994) and mutational hot-spots (Caron de Fromentel & Soussi, 1992). See the text for more details.

findings have been confirmed by the study of a mutant containing a partial deletion of block I (residues 12 to 19; Marston et al., 1994; and see Figure 1). This mutant no longer binds the mdm2 protein but retains its transactivational activity, suggesting that the overall structure of the transactivation domain rather than its sequence is important for the transactivational activity. This result explains why mutations in the transactivation domain cannot be found in human cancers. Unlike the DNA-binding region, which is highly sensitive to virtually any point mutation, the transactivation domain is sufficiently stable to be tolerant of a single mutational event. The amino-terminal region of p53 is the binding site of a large number of viral and cellular proteins, including the E1b protein from adenovirus type 5 (Braithwaite & Jenkins, 1989; Kao et al., 1990), mdm2 protein (Chen et al., 1993; Oliner et al., 1993), RPA protein (Dutta et al., 1993), which also binds at the carboxy-terminal region, hsp70 protein (Lam & Calderwood, 1992), TATA binding protein (TBP; Seto et al., 1992; Truant et al., 1993) and the transcriptional coactivators TAFII 40 and TAFII 60 (Thut et al., 1995). It should be pointed out that the amino-terminal region of p53 contains a high number of proline residues, some of them in tandem repeats. It has been suggested that such regions could function as a stiff ‘‘sticky arm’’, binding rapidly and reversibly to other proteins (Williamson, 1994). The interaction of mdm2 and p53 has been studied more closely, either by using point mutations (Lin et al., 1994) or a series of

synthetic peptides (Picksley et al., 1994). These studies enabled the definition of a very limited region (amino acid residues 18 to 23 in human p53), contained in the highly conserved block I, that includes the residues Leu22 and Trp23 shown to be important for transactivation (Lin et al., 1994). Partial deletion of block I led to the loss of mdm2 binding (Marston et al., 1994). This result therefore explains how the mdm2 protein inhibits the transactivational activity of p53 by masking the transactivation domain. These studies on transactivation and mdm2 binding and on the structure of this region raise the following considerations. The transactivation region of p53 is not specific, since it may be substituted by equivalent regions from other transcription factors (Pietenpol et al., 1994). This is not unexpected, since the same is generally true for other transcription factors. The finding that the highly conserved block I corresponds to the binding site of the mdm2 protein suggests that this conservation does not concern transactivational activity itself but rather its regulation by the specific binding of protein factor(s) such as mdm2. This is confirmed by the observation that human mdm2 binds very well to the same region of Xenopus p53 (T. Soussi, unpublished data). It should be noted that mutations that abolish mdm-2/p53 interaction are also deleterious for the binding of TAFII 40 and TAFII 60, suggesting complex regulation of the transcription domain of p53 (Thut et al., 1995). Although p53 is able to activate transcription though the binding of specific DNA regulatory

Figure 3. Alignment of p53 proteins using human p53 as the reference. Amino acid residues homologous to human p53 are indicated by hyphens. Highly conserved domains I to V are identical with those defined by Soussi et al. (1987), apart from domain I, which was extended four amino acid residues towards the carboxy terminus, as suggested by Lin et al. (1994). (*) Partial sequences. (**) The squid p53 protein contains 100 amino acids unrelated to p53 in the carboxy terminus. Unpublished sequences should not be considered until they have been published.

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sequences, it has been demonstrated that wild-type p53 specifically represses the activity of promoters whose initiation is dependent on the presence of a TATA box. Direct interaction of TBP and p53 has been largely documented (Seto et al., 1992; Liu et al., 1993; Martin et al., 1993; Truant et al., 1993; Horikoshi et al., 1995). Two p53 domains appear to be involved in this interaction; the amino-terminal region of p53 (residues 20 to 57): Liu et al., 1993; Truant et al., 1993; Horikoshi et al., 1995) and the carboxy terminus of p53 (residues 318 to 393). Both regions bind to the same region of the TBP (residues 220 to 271: Liu et al., 1993; Horikoshi et al., 1995). It has been proposed that the p53-TBP interaction could be involved in the repressive activity of p53 in transcription promoters containing a TATA box. The biological function of such activity remains to be established. The amino-terminal region of p53 is the target of phosphorylation by various kinases, one of which is particularly interesting in light of the properties of p53 (for a review, see Meek, 1994). This is a DNA-dependent kinase that phosphorylates Ser15 and Ser37 in human p53 (4 and 15 in murine p53: Lees-Miller et al., 1992; Wang & Eckhart, 1992). Mutation of Ser15 abolishes the antiproliferative ability of p53, suggesting that modification of this residue is important in the protein’s activity (Fiscella et al., 1993). The involvement of a DNAdependent kinase in the activation of p53 is intellectually stimulating because, at present, there is no firmly established link between the occurrence of a lesion in the DNA and the resulting p53 stabilization. Participation of one or several kinases that would be activated by the presence of the free end of the DNA or by the presence of small DNA fragments could explain this activation. Moreover, it has been suggested that p53 complexed with DNA is more resistant to proteolysis (Molinari & Milner, 1995). Other kinases phosphorylate the amino-terminal region of p53. Casein kinase I phosphorylates Ser4, Ser6 and Ser9 of mouse p53 (Milne et al., 1992), while the MAP kinase phosphorylates Thr73 and Thr83 of murine p53 (Milne et al., 1994). The role of these post-translational modifications remains to be elucidated. Central region of p53 The importance of this region in p53 function has been suggested by several observations: (1) the presence of four of the five evolutionarily conserved blocks (Soussi et al., 1990); (2) the high concentration of mutations in this region (Caron de Fromentel & Soussi, 1992; Greenblatt et al., 1994); and (3) the fact that it contains the binding site of SV40 T antigen (Tan et al., 1986; Jenkins et al., 1988; and see Figure 2). The finding that p53 is never mutated in SV40-transformed cells is in perfect agreement with the fact that its interaction with AgT induces its complete inactivation. It was long suggested that this central region could be the

target of certain cellular proteins like the Rb pocket, but more recent work shows that it contains the domain that binds to DNA and, more particularly, to strong p53-binding sites. The first indication of the role of the central region in DNA binding came from Halazonetis and Kandil, who demonstrated that the highly conserved evolutionary blocks IV and V were directly involved in DNA contact (Halazonetis et al., 1993). Proteolytic digestion of wild-type p53 by enzymes such as thermolysin or subtilysin generates a 27 kDa fragment containing the entire central portion of the protein (amino acid residues 92/102 to 306/292, according to the protease used: Bargonetti et al., 1993; Pavletich et al., 1993). This fragment of p53 is able to specifically bind to the RGC or CONS sequence if it comes from wild-type p53, whereas digestion fragments from mutant p53 can no longer do so (Bargonetti et al., 1993; Pavletich et al., 1993). Using truncated p53 produced in insect cells, Wang et al. (1993) defined a similar region (residues 80 to 290) necessary and sufficient for specific DNA binding. This central region of p53 may be considered as an archetypal domain, since it is a unit in terms of independent folding, protease resistance, structure, function and molecular evolution. It is noteworthy that p53 mutants are generally more stable in vivo and accumulate in the nucleus of the neoplastic cell. However, no direct evidence has been provided to show that the mutation in itself is the direct cause of p53 stabilization. Carboxy-terminal region of p53 Recent works have highlighted the importance of this region which contains at least three biological functions, i.e. nuclear localization, tetramerization and both non-specific DNA binding and recognition of primary DNA damage. Three nuclear localization signals (NLS) have been identified (Dang & Lee, 1989; Shaulsky et al., 1990; and see Figure 2). Mutagenesis of the NLS1 signal induces the synthesis of a totally cytoplasmic p53 protein, while alteration of the NLS2 or NLS3 signals leads to both cytoplasmic and nuclear localization (Shaulsky et al., 1990). The respective function of each site is currently unknown. Recently, an NLS-binding protein that interacts with p53 has been identified (Elkind et al., 1995), but its actual implication in the translocation of p53 needs to be confirmed. The wild-type p53 protein is predominantly found in the form of a tetramer. (P. Wang et al. (1994) showed that the segment of p53 consisting of amino acid residues 323 to 355 is sufficient for assembly of stable tetramers. Furthermore, the high-resolution structure of a small p53 segment (residues 319 to 360) has been studied by multidimensional NMR (Clore et al., 1994, 1995; Lee et al., 1994) and by crystal structure analysis (residues 320 to 356; Jeffrey et al., 1995). These studies confirm that residues 325 to 356 are essential for tetramerization. This domain contains

630 a b-strand (residues 326 to 333) and an a-helix (residues 335 to 354). They form a V-shaped structure with the helix axis being antiparallel to the direction of the b-strand. Gly334 is critical for the stability of this structure. Interestingly, Gly334 residues are conserved across all p53 species (Figure 3). This region forms a stable tetramer with very unusual topology, which has not been observed in other multimeric proteins. Each subunit forms a dimer via antiparallel interaction of their b-strand, and two dimers interact through hydrophobic and electrostatic contact between their a-helices to form a tetramer. Although this domain is essential for p53 activity, only a few tumor-derived mutations have been detected in the tetramerization domain. Jeffrey et al. (1995) have proposed that this domain is rather robust, and a single mutation could not interfere with oligomerization of p53. This view has been substantially confirmed by the mutational analysis performed by Waterman et al. (1995), who showed that only double or triple mutations in the tetramerization domains abolished p53 activity. The oligomerization domain is required for the negative dominant activity of p53 (Shaulian et al., 1992; P. Wang et al., 1994a). The carboxy-terminal region of p53 is able to bind non-specifically to DNA (Foord et al., 1991) but it cannot bind to the RGC or CONS sequences, for which the central region is required. Nevertheless, this region has been shown to play an important role in specific DNA binding. Indeed, specific DNA binding is very strongly activated when the 14 kDa carboxy-terminal region is phosphorylated by casein kinase II, activated by binding of a monoclonal antibody such as PAb421, or when this region is deleted by proteolysis (Hupp et al., 1992, Hupp, 1993). Small peptides derived from this negative regulatory domain have been shown to activate specific DNA binding, suggesting that this domain could block the core domain and would have to be displaced in order to permit DNA binding (Hupp et al., 1995). Recent data have shown that the carboxy-terminal region is glycosylated and could be involved in the activation of p53 (Shaw et al., 1996). Some studies have proposed a direct link between DNA damage and p53. First, Jayaraman & Prives (1995) showed that short, single-stranded DNA can stimulate sequence-specific DNA binding. This propery requires the C terminus of p53. More interestingly, a peptide spanning this region is able to stimulate this specific DNA binding in trans. Second, Lee et al. (1995) demonstrated that the C terminus of p53 recognizes DNA molecules with insertion:deletion mismatches. Lastly, it has been shown that the carboxy-terminal region has the capacity to form highly stable complexes with DNA damaged either enzymatically or by ionizing radiation. This function maps in the last 75 amino acid residues of the human and murine p53 (Reed et al., 1995). Taken together, these studies suggest a model in

Review Article: p53 Protein

which wild-type p53 could have a two-step activity after DNA lesion. In a first step, the protein could bind directly to the lesion and form a stable complex, which would lead to the accumulation of p53. Such binding induces a change in p53 conformation leading to the release of a sequencespecific competent protein. This step could be helped by post-translational modification of p53 (Hupp & Lane, 1995; Shaw et al., 1996). In the second step, this competent p53 could activate the transcription of specific genes involved in growth arrest, DNA damage or apoptosis. This hypothesis is strengthened by the identification of alternative splicing in murine and human p53 (Arai et al., 1986; Flaman et al., 1996). The major splice form retains the carboxy-terminal sequence, which regulates sequence-specific DNA binding, whereas in murine p53 the alternative splice form replaces amino acid residues 363 to 287 with 17 different residues (Arai et al., 1986). This protein fails to bind non-specifically to nucleic acids and is constitutive for sequence-specific DNA binding (Bayle et al., 1995). This region is phosphorylated by casein kinase II at the serine residue in position 392, which is highly conserved in all vertebrate p53 proteins (Meek et al., 1990; Herrmann et al., 1991; Hupp et al., 1992; and see Figure 3). In addition, this residue is the binding site of a small RNA (5.8 S rRNA), whose role is not known (Samad & Carroll, 1991). The p34 cdc2 kinase is able to form complexes, and phosphorylates human p53 at residue 315 (312 for murine p53: Addison et al., 1990; Bischoff et al., 1990; Milner et al., 1990). Given the proximity of this site to the NLS1 signal, there may be a link between this phosphorylation and transport of p53 into the nucleus, but no direct proof of this exists. The carboxy terminus of p53 is the target of several viral and cellular proteins (BZLF1, RPA and HBx); (Dutta et al., 1993; Feitelson et al., 1993; X. Wang et al., 1994) but, unlike proteins that bind to the amino terminus of p53, no biological function has been assigned to these interactions. Conformation of p53 During mitogenic stimulation of untransformed lymphocytes, p53 could be detected in two forms that reacted in a mutually exclusive manner with two monoclonal antibodies (Milner & McCornick, 1980). This observation laid the groundwork for a model described by Jo Milner proposing the existence of at least two different conformations of p53 based on their differential immunoreactivity with specific monoclonal antibodies: a ‘‘suppressor’’ conformation that can inhibit cell division (PAb240− and PAB1620+ epitopes), and a ‘‘promoter’’ conformation (PAb240 + and PAb1620− epitopes) (Milner, 1991, 1994). The equilibrium between these two forms would regulate p53 function during the cell-cycle. This hypothesis was supported by the existence of a thermosensitive mutant p53 protein from mouse (Ala : Val135; Michalovitz et al., 1990). At 37°C, this protein is

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altered in both function and conformation, while at 32°C, it exhibits wild-type behavior (Milner & Medcalf, 1990; Martinez et al., 1991). Similar behavior has been found both for the human p53 mutant CX3 (Ala : Val143; Zhang et al., 1994) and for the wild-type Xenopus p53 (Soussi et al., 1990) In the latter case, it has been demonstrated that expression of wild-type frog p53 at 37°C in mammalian cell leads to the synthesis of a protein that binds to hsp70 and is unable to transactivate a reporter gene linked to the human WAF-1 promoter. At 32°C, frog p53 behaves like authentic wild-type p53, i.e. it does not bind hsp70, and it transactivates the WAF-1 promoter. All these results suggest that p53 protein is highly flexible and that its conformation plays a key role in the regulation of its biological activity. However, in view of the crystallographic structure of p53, Cho et al. 1994 proposed that the mutant conformations instead represent a denatured state of the protein rather than well-defined distinct protein conformations. The ‘‘promoter’’ conformation defined above is generally characteristic of mutant p53s found in human cancers (Gannon et al., 1990). Some mutants are able to induce a mutant conformation to a p53 cotranslated from a wild-type gene, thereby explaining the transdominant phenomenon observed in certain cancers (see below; Milner & Medcalf, 1991). At present the mechanisms responsible for this conformational change are totally unknown. Some authors have suggested that phosphorylation of the carboxy-terminal region could be involved in this change, but more recent data indicate some involvement of phosphorylation in the amino terminus of the protein (Ullrich et al., 1992, 1993). Hainaud and Milner showed that incubation of wild-type p53 with a metal-chelating agent can induce a reversible conformational change so as to produce a protein that is ‘‘mutant’’ in terms of conformation, and in terms of DNA binding activity (Hainaut & Milner, 1993a,b). These authors postulated the presence of a zinc molecule bound to p53 via its cysteine residues. This hypothesis has been confirmed by crystallographic data (Cho et al., 1994). p53 immunogenicity in relation to its structure Monoclonal antibodies (mAbs) directed against p53 have been a major tool in most types of studies on this protein, both at the level of cloning of murine p53 (Oren & Levine, 1983), detection of various conformations (Milner, 1984; Gannon et al., 1990; Legros et al., 1994b) and immunohistochemical analysis of the protein in tumor tissues (Dowell et al., 1994). Analysis of these monoclonal antibodies has revealed that most of them recognize the linear epitopes located in the amino or carboxy-terminal regions of p53, with the exception of PAb1620 and PAb246, which recognize specific conformational epitopes of wild-type p53, and PAb240 which recognizes a cryptic epitope revealed in

mutant p53 (Stephen & Lane, 1992; Legros et al., 1994a). Production of several new batteries of monoclonal antibodies directed against wild-type and native human p53 has confirmed this phenomenon (Legros et al., 1993; Vojtesek et al., 1995). Indeed, analysis of the antibodies in sera of mice or rabbits hyperimmunized with human p53 shows that most of these antibodies recognize specific epitopes located in the amino and carboxy-terminal regions of the protein (Legros et al., 1994b). Anti-p53 antibodies are detected in sera of patients with different types of cancer (Crawford et al., 1982; Caron de Fromentel et al., 1987). Mapping of the epitopes recognized by these antibodies is fully identical with mapping results obtained in immunized animals, i.e. there exists preferential recognition of the amino- and carboxyterminal regions (Schlichtholz et al., 1992, 1994; Lubin et al., 1993). Two important conclusions may be drawn from these findings: (1) the similarity of the immune responses of both cancer patients and hyperimmunized animals suggests that it is overexpression of the p53 protein in tumor cells that determines the immune response of the patients; (2) the amino (residues 1 to roughly 95) and carboxy-terminal (residues 300 to 393) regions of p53 are highly exposed and accessible at the protein surface, while the central region seems to be buried in the interior of the molecule. However, using a special selection strategy, eight mAbs directed against the central region of the molecule and thus defining four new epitopes have been obtained (Legros et al., 1994b). Three-dimensional structure of p53 and conserved blocks X-ray crystallography of human p53 has been an important step in the understanding of the structure of this protein. The central region (amino acid residues 102 to 292) has been crystallized in the form of a protein-DNA complex (Cho et al., 1994). This core region has been shown to include the following motifs: (1) two antiparallel b-sheets composed of four and five b-strands, respectively (Figures 1 and 2). These two sheets form a kind of compact sandwich that holds the other elements; (2) a loop-sheet-helix motif (LSH) containing three b-strands, an a-helix and the L1 loop; (3) an L2 loop containing a small helix; and (4) an L3 loop composed mainly of turns. It is quite remarkable to note the striking correspondence between these various structural elements and the four evolutionarily conserved blocks (II to V). The LSH motif and the L3 helix are involved in direct DNA interaction (LSH with the major groove and L3 with the minor groove). The L2 loop is presumed to provide stabilization by associating with the L3 loop. These two loops are held together by a zinc atom tetracoordinated to Cys176 and His179 on the L2 loop, and Cys278 and Cys242 on the L3 loop. Mutational analysis shows that these three conserved cysteine residues are essential for

632 DNA binding and transactivation (Rainwater et al., 1995). Analysis of the distribution of mutations in p53 shows that they are essentially clustered in the central region of the protein, and especially in the four blocks II to V, which have been identified as the DNA-binding region. In view of the three-dimensional structure of the protein, it has been proposed that two classes of mutations can be predicted: class I mutations, which affect amino acid residues directly involved in the protein-DNA interaction (residues in the LSH and L3), and class II mutations, which affect amino acid residues involved in stabilization of the three-dimensional structure of the protein (residues in L2). In fact, it has long been established that mutant p53 could undergo conformational changes leading to its interaction with the heat shock protein hsp70, but also modifying either positively or negatively the accessibility to certain monoclonal antibodies such as PAb1620, which recognizes a specific conformational epitope of wild-type p53, or PAb240, which recognizes a cryptic epitope revealed in mutant p53. As mentioned above, the situation is not simple, and there have been various reports of a certain degree of diversity in the behavior of different p53 mutants. In particular, some p53 mutants have been shown to be PAb1620 + /PAb240 − /hsp70 − . With the aim of analyzing a possible correlation between conformational changes and loss of activity, Ory et al. (1994) studied a library of 23 p53 proteins mutated in the three hot-spot codons, Arg175, Arg248 and Arg273. The results show that these mutants may be classified into two different phenotypes, based on the reactivities with hsp70 and with monoclonal antibodies PAb1620 and PAb240. Phenotype PAb1620−/PAb240+/hsp70+ corresponds to all mutations found in codon 175 and to a single mutant in codon 273 (Arg : Pro), while phenotype PAb1620+/PAb240−/hsp70− corresponds to mutations in codons 248 and 273 (Ory et al., 1994). No intermediate case was found and each of these mutant p53 proteins had lost its transactivation and growth inhibition activities. Furthermore, the conformational changes in p53 were dissected by a new battery of monoclonal antibodies directed against the central region of the protein (Legros et al., 1994b). Like PAb240, none of these antibodies was able to recognize native, wild-type p53. On the other hand, regardless of the location of their epitopes, they were all able to recognize p53 mutants that had undergone conformational changes (Legros et al., 1994b). This result indicates that the central region of wild-type p53 protein has a very compact structure, which is held in place by the two antiparallel b-sheets. Deleterious mutations that do not directly affect the DNA-binding site cause this compact structure to loosen and/or misfold, thus revealing these cryptic epitopes. Characterization of these two classes of mutations emphasizes that p53 alterations are highly heterogeneous. First, it has been shown that various p53

Review Article: p53 Protein

mutants have a behavior that is cell-type-dependent (Forrester et al., 1995). Second, clinical data show that some mutations are associated with a worse prognosis than other mutations (Bergh et al., 1995; Goh et al., 1995).

Conclusion In 1990, we proposed a model for the structural organization of p53 that divided this protein into three distinct regions on the basis of charge, hydrophobicity and the presence of conserved domains (Soussi et al., 1990). Recent data have confirmed the great usefulness of this model at both the functional and structural levels (Pavletich et al., 1993). Further insight into the structure and function of the various p53 mutants has revealed that not all p53 mutants are equivalent, and may be classified into two classes (Ory et al., 1994). Class I mutants correspond to residues involved in the stabilization of the tertiary structure of the p53, whereas class II mutants correspond to DNA contact mutants that affect p53 residues directly involved in DNA binding. This knowledge is of fundamental importance, not only for satisfying our curiosity about this fascinating protein, but also with respect to clinical considerations that concern more than 50% of patients with various types of cancer. Indeed, although direct gene therapy by introduction of a normal p53 gene into tumor cells does not yet seem feasible, the possibility of ‘‘rehabilitating’’ the mutant protein itself may not be an elusive goal. Considering the data described above showing the extent to which mutations can affect the three-dimensional structure of the protein, the design of molecules to specifically target the mutant protein is perfectly conceivable. These molecules could either induce tumor cells to undergo apoptosis or render them more sensitive to various chemotherapeutic agents.

Acknowledgements We thank C. Caron de Fromentel, P. Devilee, S. Friend, E. May, R. Nairn, V. Notario, M. Okuda and P. Winge for communicating their results prior to publication. This work was supported by grants from the Association de la Recherche sur le Cancer and the Ligue Nationale contre le Cancer (Comite´ de Paris and Comite´ National).

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Review Article: p53 Protein

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Review Article: p53 Protein

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Review Article: p53 Protein

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Review Article: p53 Protein

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Review Article: p53 Protein

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Edited by M. Yaniv (Received 29 September 1995; received in revised form 24 April 1996; accepted 1 May 1996)