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Oncogene (1999) 18, 2405 ± 2410 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc SHORT REPORT De®nition of a p53 transactivation function-de®cient mutant and characterization of two independent p53 transactivation subdomains Corinne Venot1, Michel Maratrat1, VeÂronique Sierra1, Emmanuel Conseiller1 and Laurent Debussche*,1 1 Centre de Recherche de Vitry-Alfortville, RhoÃne-Poulenc Rorer, 13 quai Jules Guesde, 94403 Vitry sur Seine Cedex, France The wild-type protein product of the p53 tumor suppressor gene can activate transcription of genes which are involved in mediating either growth arrest, e.g. WAF1 or apoptotis, e.g. BAX and PIG3. Additionally, p53 can repress a variety of promoters, which, in turn, may be responsible for the functional activities exhibited by p53. This study shows that the Q22, S23 double mutation, which is known to inactivate a p53 transactivation subdomain located within the initial 40 residues of the protein, while abrogating transactivation from the WAF1 promoter, only attenuates apoptosis triggering, transactivation from other p53-responsive promoters and repression of promoters by p53. The Q53, S54 double mutation, which inactivates another p53 transactivation subdomain situated between amino acids 43 and 73 results in attenuation of all of the aforementioned p53 activities. In contrast to the Q22, S23 double mutation, this latter mutation set does not alter mdm-2-mediated inhibition and degradation of p53. Finally, mutation of all four residues results in complete abrogation of every p53 activity mentioned above. Keywords: p53; apoptosis; transactivation; repression; PIG3 p53 is a critical tumor suppressor gene, which can respond to multiple signals of cellular alarm, by inducing either growth arrest or apoptosis (reviewed in Bates and Vousden, 1996; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997). p53 protein is a transcriptional transactivator and induces speci®c target genes by binding to short genomic sequences, called p53-responsive elements (p53RE), which ®t a consensus sequence to variable extents (Funk et al., 1992; El-Deiry et al., 1992). Sequence speci®c transactivation (SST) by p53 has been shown to be dependent upon three major independent domains: (i) a central core domain responsible for sequence-speci®c DNA binding (amino acid residues 102 ± 292) (Bargonetti et al., 1993; Pavletich et al., 1993; Cho et al., 1994); (ii) a Nterminal transactivation domain responsible for recruitment of transcription machinery (amino acid residues 1 ± 40) (Unger et al., 1992; Lin et al., 1994; Thut et al., 1995; Lu and Levine, 1995); and (iii) a C-terminal *Correspondence: L Debussche Received 23 June 1998; revised 28 October 1998; accepted 29 October 1998 oligomerization domain (amino acid residues 319 ± 360) (Sturzbecher et al., 1992; Clore et al., 1994; Jerey et al., 1995). The importance of SST in p53 tumor suppressor activity is underscored by the fact that the vast majority of p53 mutations derived from tumors map within the sequence-speci®c DNA binding domain (Hollstein et al., 1991; Pavletich et al., 1993; Cho et al., 1994). Growth arrest by p53 seems clearly due to SST of several p53 target genes, such as WAF1, GADD45 and cyclin G (reviewed in Bates and Vousden, 1996; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997). However, many reports suggest that p53 can mediate apoptosis through either transcriptional regulation-dependent or independent pathways. The transcriptional regulation-dependent pathway involves induction by p53 of a growing list of p53 target genes which are involved in apoptosis triggering, such as BAX (Miyashita and Reed, 1995), FAS/APO1 (Owen-Schaub et al., 1995), IGF-BP3 (Buckbinder et al., 1995), Cathepsin D (Wu et al., 1998), Killer/DR5 (Wu et al., 1997), PAG608 (Israeli et al., 1997) and PIG3 (Polyak et al., 1997). On the other hand, the transcriptional regulation-independent pathway remains largely obscure (Caelles et al., 1994; Wagner et al., 1994) and much of the evidence describing its existence stems from studies with SSTde®cient p53 mutants, most especially the p53 transactivation domain mutant Q22, S23 (M22/23) which showed them to be still capable of inducing apoptosis in certain tumor-derived cell lines (YonishRouach et al., 1994; Haupt et al., 1995, 1996; Chen et al., 1996). Firstly, we con®rmed that M22/23 retains growth suppression and apoptotic activities in tumor cells. In a colony formation assay performed with H1299 (Figure 1) and Saos-2 (data not shown) cell lines, which are both p537/7, M22/23 displays a phenotype which is intermediate between the inactive tumor-derived mutant, D281G, and p53 wild-type (p53 WT). This assay enables growth suppression in its most general sense to be measured as it scores for either p53dependent growth arrest and/or apoptosis in tumor cells that receive the p53 protein. In the case of M22/ 23, the remaining growth suppression eect results in great part from apoptosis as this mutant retains signi®cant apoptotic activity (Figure 2, columns 1 ± 3) but no detectable growth arrest activity (data not shown), which is consistent with previous reports (Chen et al., 1996, Walker and Levine, 1996). Recent studies have demonstrated the existence of functionally dierent p53RE, which could be classi®ed according to their anities for p53 WT and certain non-apoptotic p53 mutants (Rowan et al., 1996; Characterization of p53 transactivation domains C Venot et al 2406 Figure 1 M22/23 and M53/54 retains growth suppression activity while p53QM was completely lost it. p537/7 human lung carcinoma cell line H1299 were seeded at 1.5 105 cells per multi-well six culture testplate and transfected with 1.5 mg of p53 expression plasmid (upper panel) or 0.75 mg of p53 expression plasmid+0.75 mg of pcDNA3 empty vector (lower panel), preincubated for 30 min with 5 ml lipofectAMINE reagent (GIBCO ± BRL, Gaithersburg, MD, USA). Selection of drug-resistant colonies was performed as follows: 24 h after transfection, cells were seeded in 10-cm plates, cultured in 400 mg/ml G418 containing medium for 2 ± 3 weeks and then colored with fuchsin Figure 2 FACS analysis of apoptotic activity of p53 mutants. Cells were analysed as described previously (Conseiller et al., 1998) with some modi®cations. Brie¯y, 48 h after transfection, supernatants were collected and anchored cells were trypsinized. Floating and trypsinized cells were combined, ®xed in 1% formalin and permeabilized with Triton 0.1%. They were then incubated with PAb 240 antibody (which recognized both wild type and mutants p53 under our denaturating ®xation conditions, data not shown) and subsequently with Goat anti-mouse FITC (Immunotech, Marseille, France). They were ®nally counterstained with 10 mg/ml Propidium iodide (PI) in 1 mg/ml boiled Rnase and analysed on a FACS Epics Elite ESP II (Coulter, Miami, FL, USA). Transfection eciencies were evaluated as the percentages of positive cells for PAb 240. In each independent experiment, percentages were very similar for all ®ve p53 proteins and ranged between 30 and 60% from one to another experiment. Percentage of apoptotic cells were quanti®ed on the basis of their positivity for Pab 240 and their relative DNA content (sub G1). This ®gure is a compilation of three independent experiments Ludwig et al., 1996; Friedlander et al., 1996; Venot et al., 1998). We thus reinvestigated SST by M22/23 on a larger set of promoters than the ones used by others to establish its SST-de®ciency. By transient transfection experiments using luciferase reporter constructs (Figure 3, columns 1 ± 3), we ®rstly showed that M22/23 was unable to transactivate a construct containing the WAF1 promoter. This inactivity was observed at optimal plasmid concentration as indicated in the titration curve of p53 WT inputs inserted in the upper right corner of Figure 3, but also at largely higher M22/23 plasmid concentrations (data not shown). On the other hand, M22/23 induced a low but signi®cant expression of reporter gene from the BAX, MDM2 and, interestingly, PIG3 promoters. Transactivation from this last promoter is strongly correlated with p53dependent apoptosis in colon and lung cancer cells and, especially, in H1299 cells (Polyak et al., 1997; Venot et al., 1998). These results indicate that apoptosis by M22/23 is most probably SST-dependent, since this mutant is not totally de®cient but rather attenuated in its ability to induce p53-responsive apoptotic genes. This remaining SST activity could be due to the second transactivation subdomain within p53 N-terminal domain. This second subdomain, called subdomain 2, has been localized to a region encompassing residues 43 ± 73 by deletion analysis (Chang et al., 1995) and functionally characterized in yeast (Candau et al., 1997). Within subdomain 2, two hydrophobic residues W53 and F54 play a critical role, suggesting thus that it is structurally similar to both VP16 and the ®rst p53 transactivation domains (Cress and Treizenberg, 1991; Regier et al., 1993; Lin et al., 1994; Candau et al., 1997). We therefore mutated both residues W53 and F54 together in p53 WT and M22/ 23, in order to generate the mutant Q53, S54 (M53/54) or the quadruple mutant at positions 22, 23, 53 and 54 (p53QM), respectively. These mutants were evaluated in the three above-mentioned functional assays: colony formation assay, apoptosis and transactivation of dierent functional promoters. As shown in Figure 1, p53QM has totally lost growth suppression activity and this is correlated with a corresponding loss of apoptotic Characterization of p53 transactivation domains C Venot et al activity (Figure 2, column 4). Moreover, this mutant is completely de®cient for transactivation of all promoters tested (Figure 3, column 4). This mutant can be thus de®ned as a p53 transactivation function-de®cient mutant. On the other hand, functional characterization of M53/54 revealed that it displays a phenotype which is intermediate to that displayed by p53 WT and M22/23. It exhibits slightly stronger growth suppression and apoptotic activities than M22/23 (Figures 1 and 2). In transactivation assays, a similar quantitative dierence was observed when the MDM2, BAX and PIG3 promoters were used. Results obtained with the WAF1 promoter are consistent with the above- mentioned inactivity of M22/23 on this promoter, since the activation due to M53/54 is nearly identical to the activation due to p53 WT. This led us to conclude that transactivation subdomain 1 but not subdomain 2 is critical for p53-dependent activation of the WAF1 promoter. A functional dierence between both subdomains is not only observed with WAF1 promoter activation. As shown by transient cotransfection with a MDM2 expressing vector (Figure 4a), only transactivation by subdomain 1 is sensitive to MDM2-mediated inactivation, thus con®rming previous reports (Lin et al., 1994; Candau et al., 1997). As expected and shown in Figure 4b, mutations in subdomain 2 do not aect the recently described Figure 3 Transcriptional activity of p53 mutants on various promoters. The transcriptional activity was measured at optimal plasmid concentration as indicated in the titration curves of p53 WT inputs inserted in the upper right corner of each barchart. 0.3 mg of luciferase reporter gene under the control of dierent p53 speci®c promoters and 0.1 mg of p53 expression vectors were used in each transfection. For titration curves, p53 WT plasmid concentrations used were 1, 3, 10, 30, 100 and 300 ng. The concentration used for the experiments (0.1 mg) is indicated by the arrow. Twenty-four hour after transfection, H1299 cells were rinsed with phosphate-buered saline (PBS), resuspended in cell lysis buer (Promega), and incubated for 20 min at room temperature. Samples were centrifuged and luciferase activity of the cleared supernatant was determined in the presence of luciferin (Promega) and ATP. Each experiment was repeated at least three times to calculate the means and standard deviations 2407 Characterization of p53 transactivation domains C Venot et al 2408 Figure 4 (a) MDM2 can reduce SST by p53 WT and p53 M53/54 on PIG3 promoter but not SST by p53 M22/23. H1299 cells were transiently transfected with 0.3 mg of PIG3 promoter (upstream from the luciferase gene), 0.1 mg of p53 expression vectors and in presence or absence of the MDM2 expression plasmid (Dubs-Poterszman et al., 1995). Luciferase assay was performed as described in Figure 3. (b) Expression levels of p53 mutant proteins in absence or presence of MDM2 protein. H1299 cells were transiently co-transfected with 1 mg of p53 mutant expression vectors and with either MDM2 expression vector or empty vector. Cells (56105) were collected 24 h after transfection and lysed for 15 min at 48C with 100 ml of RIPA buer and centrifuged at 15 000 r.p.m. for 15 min. The supernatants were collected, 1/3 loaded onto 10% SDS ± PAGE gels, and blotted onto PVDF membranes (New England Nuclear, Boston, MA, USA). p53 WT and p53 mutants were detected with Pab240 anti-p53 monoclonal antibody and with anti-mouse horseradish peroxidase-conjugated secondary antibody. Grb2 protein content was used as loading control by using anti-grb2 monoclonal antibody (Transduction Laboratories) and anti-mouse horseradish peroxidase-conjugated secondary antibody, after SDS ± PAGE migration of samples on 14% gels. Proteins were visualized by chemiluminescent detection (ECL) (Amersham) MDM2-mediated degradation of p53 (Haupt et al., 1997; Kubbutat et al., 1997). Besides it appeared that M53/54 and mainly D281G were more strongly degraded by MDM2 than p53 WT. Both amino-terminal and C-terminal regions of p53 have been shown to be involved in repression by p53 (reviewed in Gottlieb and Oren, 1996; Ko and Prives, 1996). This activity may be in¯uential in apoptosis triggering (Murphy et al., 1996; Venot et al., 1998 and references therein). Within the amino-terminal region, both subdomain 1 and proline-rich domain of p53 have been shown to be required for the repression function of p53 (Roemer and Mueller-Lantzsch, 1996; Venot et al., 1998). We have also investigated the potential role of subdomain 2 in mediating repression by using M53/ 54 and p53QM in transient co-transfection experiments Characterization of p53 transactivation domains C Venot et al with a RSV LTR promoter construct. Results presented in Figure 5 show that p53QM, like the tumor-derived p53 mutant D281G, is unable to repress RSV LTR promoter. On the other hand, M53/54, like M22/23, retains partial repression activity on RSV LTR. This led us to conclude that both transactivation subdomains are required for full repression of promoters by p53. The 12 residue overlap between the end of subdomain 2 and the start of the so-called prolinerich domain which has been localized between residues 62 and 91 (Walker and Levine, 1996) raises the question of possible functional interdependence between the two regions. In fact, these domains seem to be totally independent, since inactivating mutations of either domain cause opposite phenotypes linked to apoptotic activity. Whereas the proline-rich domaindeletion mutant is totally silent for transactivation of PIG3 promoter, has lost repression activity, and is de®cient for apoptosis triggering (Venot et al., 1998), M53/54 retains signi®cant levels of all three activities (this study). This, in turn, shows that the phenotype displayed by the proline-rich domain-deletion mutant is not attributable to the impairment of any transactivation function of p53 subdomain 2, e.g. on the PIG3 promoter. At this stage, we can conclude that the region encompassing the ®rst hundred residues of p53 contains three independent functional domains: transactivation subdomain 1 from residues 1 ± 40, transactivation subdomain 2 from 43 ± 73, and proline-rich domain from 62 ± 91. The proline-rich domain mediates some cellular activation of DNA binding on low anity p53RE, e.g. PIG3 promoter and is also required for repression of promoters (Venot et al., 1998). Subdomains 1 and 2 cooperate with respect to the transcriptional regulation activities of p53, i.e. SST and repression of promoters, subdomain 1 being more critical for transactivation of WAF1. Transactivation and repression by p53 may operate through direct contacts with components of the general transcription machinery (reviewed in Bates and Vousden, 1996; Gottlieb and Oren, 1996; Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997). It would be of utmost interest to study the speci®city of each subdomain for these components. Importantly, in vivo studies in yeast have shown that the two subdomains require the yeast adaptor complex ADA2/ADA3/GCN5 to varying extents (Candau et al., 1997). The observation that both M22/23 and M53/54 but not p53QM retain apoptotic activity, does not favor the hypothesis that either transactivation subdomain would be a speci®c docking site for proteins involved in transcriptional regulation-independent apoptosis by p53. We thus infer that apoptosis by p53 in H1299 2409 Figure 5 Repression activity of p53 mutants. 0.3 mg of plasmid containing luciferase reporter gene under the control of RSV LTR promoter (Venot et al., 1998) and 0.1 mg of p53 expression vectors were used in each transfection. Luciferase assay was performed as described in Figure 3. The transcriptional activity was measured at optimal plasmid concentration (0.1 mg) as indicated by the arrow in the titration curves of p53 WT inputs inserted in the upper left corner of this barchart. 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