The role of the C' terminus of murine p53 in the p53/mdm-2 regulatory loop

Nava Almog, Michael Milyavsky, Perry Stambolsky, Ayellet Falcovitz, Naomi Goldfinger and Varda Rotter,1

Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot 76100, Israel


    Abstract
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 Abstract
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 References
 
Mdm-2 plays a central role in the regulation of p53 protein level and activity. Although the interaction of mdm-2 and p53 occurs through the N-terminus of the p53 protein, our present data suggest that the C' terminus plays an important role in the regulation of the p53/mdm-2 loop. Comparative analysis of the murine regularly spliced form of p53 (RSp53) and a physiological C-terminally modified p53 protein, which results from alternative splicing of the p53 mRNA (ASp53), indicated that the two isoforms behave differently in the p53/mdm-2 loop. We found that ASp53 can preferentially induce higher levels of the mdm-2 protein, compared with RSp53. Although the transactivation capacity of both forms is inhibited by mdm-2, only RSp53 is directed to proteolytic degradation by mdm-2, while ASp53 is relatively resistant. We present evidence that suggests that ASp53 protein levels determine the biological activities mediated by RSp53, such as the induction of apoptosis, through the mdm-2/p53 regulatory loop. We suggest, therefore, a new mechanism for the regulation of p53, and show that alteration of the p53 extreme C' terminus can significantly change the transcription activity and the resistance to degradation properties of the p53 protein.


    Introduction
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 Introduction
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The p53 tumor suppressor protein plays a major role in the regulation of tissue homeostasis and in the prevention of genetically damaged cell proliferation. The most pronounced activity of p53 is its ability to suppress tumor development, which correlates with its capacity to induce apoptosis, to specifically bind DNA and to induce transactivation of down stream genes. Different stress signals, such as DNA damage, hypoxia, metabolic changes and activated oncogenes were shown to activate wild-type p53 in normal tissues (for review see refs 1 and 2).

One of the main regulators of p53 protein level and activity is the mdm-2 protein. Mdm-2 was shown to bind p53, inhibit its transactivation capacity and direct it to proteolytic degradation (for review see refs 3 and 4). Indeed, mdm-2 was shown to target p53 ubiquitination (5) and to function as a ubiquitin ligase (6). Furthermore, the mdm-2 gene is one of the p53 target genes whose transcription is induced by p53. Taken together, a negative autoregulatory loop exists between p53 and mdm-2. This feedback loop is responsible for the maintenance of the basal low levels of p53 in normal conditions. The inhibitory effect of mdm-2 can be modulated by multiple mechanisms. These include post-translational modifications, occurring both on p53 and mdm-2 proteins and physical interactions with other cellular proteins, such as p19ARF and c-abl (for review see ref. 2).

Another determinant controlling p53 protein activity and stability is the p53 C' terminus. The C' terminus of p53 contains a number of structural domains, among which are the oligomerization domain, three nuclear localization signals (NLS) (7) and nuclear export sequences (NES) (8). It also contains a number of sites for a variety of post-translational modifications, which include phosphorylation (9), acetylation (10), glycosylation (11), poly ADP-ribosylation (12), ubiquitination (13) and sites for proteolytic cleavages (14). There also exists a remarkable number of binding sites to cellular proteins, such as the TATA-binding protein (TBP) (15), several transcription factor IIH (TFIIH)-associated factors (16), the 14-3-3 proteins (17), BRCA1 (18), the S100B calcium binding protein (19), the Werner syndrome protein (20), Ref-1 (21), telomerase (22) and Sumo-1 (23). The majority of these modifications and interactions with other cellular proteins are implicated in the regulation of the p53 biological functions. Indeed, the p53 C' terminus was shown to play a major regulatory role in the ability of p53 to specifically bind DNA (2428), which is one of the hallmarks of wild-type p53, and to be essential for the recognition of and binding to damaged DNA (29,30).

Although mdm-2 binds p53 within its transactivation domain, located at the N-terminus of the protein, several findings suggest that the p53 C' terminus might determine the sensitivity of p53 to proteolytic degradation. Mutation of lysine residues within the p53 C' terminus resulted in resistance to E6-mediated degradation (31). Deletion of this domain stabilized the truncated protein (13,32), that resulted in resistance to mdm-2 directed degradation. Furthermore, the ubiquitin-like protein, Sumo-1, was shown to bind the p53 C' terminus, on lysine 386, leading to p53 activation (23,33). The c-abl protein kinase was also shown to directly bind to the extreme C' terminus of p53, which in turn enhanced its transcriptional activity and increased the p53 expression level, through the inhibition of the mdm-2 mediated degradation of p53 (34,35).

The physiological importance of the C' terminus is evident by the existence of a physiological variant of the p53 protein which results from alternative splicing of the murine p53 mRNA (ASp53). ASp53 lacks 26 amino acids from the extreme C' terminus of the p53 regularly spliced form (RSp53), and contains instead 17 new and unique amino acids (36). It consists of 25–33% of the total p53 mRNA in the cell (37,38). Moreover, the ratio between the RSp53 and ASp53 protein levels vary according to the state of the cell (e.g. proliferating or differentiating cell, and depending on the stage of cellular differentiation, and on the cell cycle phase). Interestingly, differences in the RSp53 and ASp53 pattern of expression were also monitored during tumor development and progression (39), suggesting a strong selection against ASp53 expression during tumor development. Although the murine ASp53 is relatively abundant, an ambiguity exists regarding the biological role of this spliced variant. A significant difference exists between the two physiological p53 variants in their ability to spontaneously bind specific DNA sequences (2426), and in the capacity to catalyse re-annealing of single-stranded RNA or DNA (40). Moreover, we previously showed that although the two forms are capable of inducing apoptosis when equally expressed in myeloid cells, apoptosis induced by ASp53 is attenuated, in comparison to that induced by RSp53 (41). Surprisingly, the co-expression of the two forms resulted in the inhibition of RSp53-induced apoptosis, thus suggesting a role for p53 mRNA alternative splicing in the determination of p53-induced apoptotic levels (42).

In order to further elucidate the role of the p53 C' terminus in protein stability associated with the mdm-2 degradation regulatory loop, we analysed the relationship between the two p53 alternative spliced forms and the mdm-2 protein. We found that while RSp53 was more efficient in the induction of transactivation of all of the p53 target genes examined, ASp53 selectively induced a stronger transactivation of mdm-2. Moreover, although both of the p53 protein forms seem to physically associate with the mdm-2 protein and their transcription activity was inhibited by mdm-2, there was a marked difference in their sensitivity to mdm-2 directed degradation. RSp53 protein was sensitive to the proteolytic degradation regulated by mdm-2 while ASp53 protein was relatively resistant. This suggests that by facilitating the selective degradation of RSp53, ASp53 may modulate the biological functions exerted by RSp53. Thus, alternative splicing of p53 mRNA may affect the RSp53 protein stability and activity.


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Cell lines and transfections
Murine 174.2 cell line was kindly provided by G.Lozano. The cell line was established from p53–/–mdm2–/– embryos (43). Murine K/O cell line was kindly provided by L.Donehower. Both of these cell lines were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% FCS and antibiotics. The cell line was established from p53–/– embryos. Human H1299 non-small cell lung carcinoma cell line was kindly provided from M.Oren. These cells are p53-null and are maintained in RPMI 1640 medium with 10% fetal calf serum. 174.2 and K/O cell lines were transiently transfected by LipofectAMINE reagent (Life Technologies, Gaithersburg, MD). H1299 cell line was transfected by the calcium phosphate method as previously described (42).

Plasmids and antibodies
The reporter plasmids containing the luciferase gene downstream to different p53-responsive promoters, as well as wild-type mdm-2 and p19 ARF coding plasmids, were kindly provided by M.Oren. The GADD45-luc plasmid was kindly provided by C.Prives. RSp53 and ASp53 cDNA were cloned under the constitutive promoter of CMV. The antibodies that were used were as previously described (41). pAb-248 anti-p53 monoclonal antibodies were used. pAb-4B2 anti-mdm-2 were used in the experiments.

Protein analysis and transactivation assay
Cells (0.5x106) were collected, lysed in sample buffer (140 mM Tris pH 6.8, 22.4% glycerol, 6% SDS, 10% ß-mercaptoethanol, 0.02% bromophenol blue) and subjected to SDS–PAGE. The proteins were detected using the Protoblot Western Blot Ap System (Promega, Madison, WI). The nitrocellulose membranes were blocked with 5% dry skimmed milk in TBST (10 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20) for 30 min. Incubation with the first antibody for 30 min was followed by three washes with TBST, and incubation with a second antibody conjugated to HRP enzyme for 30 min. The staining was observed using the enhanced chemiluminescence protocol (Amersham, Arlington Heights, IL).

For luciferase assays, 174.2 cells were seeded in 24-well plates and each transfection was done in triplicate. p53 coding plasmids (1 ng) and 1 µg reporter plasmid were co-transfected together with an empty plasmid for the maintenance of constant amount of transfected DNA. Twenty-four hours following transfection the luciferase activity was determined as previously described (42).

Cell viability assay
Apoptosis of transiently transfected cells was analysed by PI staining. The cells were transfected with a constant amount of total DNA which includes the p53 coding plasmids together with a GFP coding plasmid and an empty pCMV plasmid. The cells were collected 48 or 72 h post-transfection and washed in cold PBS. Cells were fixed in 70% methanol in HBSS and incubated overnight at –20°C. Cells were washed twice in PBS and incubated with 50 µg/ml RNase A and 25 µg/ml PI. Samples were then analysed in a cell sorter (FACSORT, Becton Dickinson). Highly GFP expressing cells were gated and thus the cell cycle pattern of only the highly GFP-positive cells fraction was analysed according to their PI staining. Apoptotic cells were statistically calculated according to the percentage of the sub-G1 fraction.


    Results
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 Abstract
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 Results
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 References
 
Preferential induction of mdm-2 by ASp53
In the first experiment we compared the transactivation capabilities of the two p53 physiological variants. A series of reporter constructs containing the firefly luciferase gene under the control of different p53-downstream promoters were used. In order to examine the inter-relationship of the two p53 protein forms with mdm-2, we utilized the 174.2 murine cell line, which was established from p53/mdm-2 double knock-out embryos (43). These cells were transiently transfected with the luciferase reporter plasmid together with equal amounts of plasmids encoding for either RSp53, ASp53 or an empty vector as a control (Figure 1AGo). All of the examined promoters, except for the mdm-2 promoters, were strongly activated by RSp53, while ASp53 induced significantly lower levels of transactivation. In the case of the Bax and Waf-1 promoters, the transactivation by ASp53 reached as low as 50% of that observed with RSp53. These differences are in agreement with a previous report (44).




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Fig. 1. Comparison of the induction of p53-downstream gene expression by RSp53 and ASp53. (A) Luciferase assays were performed in 174.2 p53/mdm-2 double knock-out murine cells. The cells were transiently transfected with a constant amount of reporter luciferase plasmid, carrying the luciferase gene under the control of a variety of p53 downstream promoters. Equal amounts of expression plasmids, which contain either RSp53 or ASp53 cDNA, or an empty vector as a negative control, as indicated for each experiment, were co-transfected with the reporter plasmid. The luciferase activity in each transfection was calculated according to the transfection efficiency, which was determined by the ß-gal activity. (B) The induction of mdm-2 expression was analysed by western blotting of the murine stable M1/2-derived clones: p53tsAS-27, which expresses the ASp53 protein, and clone p53tsRS-53, which expresses the RSp53 protein. pLXSN p53 null cell line was used as a control. The cells were transferred to 32°C for different time periods, as indicated in the figure, and then collected and analysed on SDS–PAGE gel. The expression of mdm-2 was analysed by the pAb-4B2 anti-mdm2 monoclonal antibody. p53 protein levels were analysed by the antibody Pab-248 anti p53 monoclonal antibodies. The expression of {alpha}-tubulin was analysed, as a control for protein level in each sample, using a monoclonal antibody directed to the {alpha}-tubulin protein.

 
Interestingly, however, this pattern was different in the case of the mdm-2 promoters. The mdm-2 gene contains two distinct promoters: an upstream constitutive promoter (P1), whose activity is barely affected by p53, and another promoter (P2), which is located in intron 1 and is p53-dependent (45). In contrast to all of the other examined promoters, only the transcription level induced by ASp53 from the mdm-2 promoters was shown to be significantly higher than that induced by RSp53 (Figure 1AGo). This was mostly pronounced with the P2 p53-dependent promoter, but was also evident with the P1 constitutive promoter (note the differences in the scales of the y-axis) and both of the promoters together (P1 + P2).

In order to validate the relevance of the above findings, we investigated the pattern of mdm-2 protein induction in murine myeloid p53-null M1/2 cells. Previously we generated and characterized M1/2-derived stable clones which express similar levels of exogenous temperature sensitive (ts) mutants of either RSp53 or ASp53 (41). For the next experiment we used the stable clones p53tsRS-53, which expressed RSp53, and the stable clone p53tsAS-27, which expressed ASp53, as well as the clone pLXSN, which was infected by an empty virus that serves as a control (41). All clones were transferred to 32°C to permit the expression of the wild-type p53 protein conformation. Cells were collected at different time points following the temperature shift, and the protein level of mdm-2 was determined using western blot. As can be seen in Figure 1BGo, a clear induction of mdm-2 was observed in both p53 expressing clones. Clone pLXSN exhibited a low basal level of mdm-2 protein induction with no significant induction. It should be noted, however, that induction of the mdm-2 expression was more pronounced in the p53tsAS-27 clone, compared with that in clone p53tsRS-53 (Figure 1BGo). Furthermore, in this time period, there was no significant change in the protein level of the p53 protein variants. Therefore, as can be seen in different and independent experimental systems, ASp53 can up-regulate mdm-2 expression more efficiently than RSp53.

It should be noted that previously we found by RT–PCR, Northern and western blots that the induction of p53 downstream genes (Waf-1, Cyclin G and Bax) is faster and more efficient by RSp53 as compared with that by ASp53 (41). These results further support the above findings, which show a selective and preferential induction of mdm-2 by ASp53.

A differential sensitivity of RSp53 and ASp53 to mdm-2 directed degradation
Previously it was shown that the extreme C-terminus of p53 is required for efficient mdm-2 directed degradation of p53, and that its deletion stabilizes the protein (32). Since RSp53 and ASp53 differ at their C' terminus, we compared their sensitivity to proteolytic degradation. For that purpose we transiently co-transfected murine 174.2 p53/mdm-2 double knock-out cells, with RSp53 or ASp53 expression plasmids, together or without a wild type mdm-2 expression vector. In agreement with previous reports (46,47) we found that upon expression of mdm-2 a significant reduction in the RSp53 protein level was evident (Figure 2AGo). In contrast, mdm-2 expression did not affect significantly the ASp53 protein levels under the same experimental conditions. It should be noted that the mdm-2 protein level is similar in cell transfected with either RSp53 or ASp53. In all transfections, the same level of total protein was analysed (shown by {alpha}-tubulin antibody staining), and similar efficiencies of transfection were achieved.



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Fig. 2. Analysis of mdm-2 directed degradation of p53 protein variants. (A) Western blot of p53 protein variants' expression using the pAb 248 monoclonal antibody. Constant amounts of the p53 protein variants coding plasmid was transfected with increasing amounts of mdm-2 expression plasmid, into 174.2 p53/mdm-2 double knock-out cells, as indicated in the figure. Mdm-2 protein level was analysed by pAb-4B2 monoclonal antibody and p53 was analysed by pAb-248. Equal amounts of analysed protein is shown by the expression of {alpha}-tubulin. (B) Western blot of the p53 protein variants' expression following transient transfection into K/O p53-null cells, using the pAb 248 monoclonal antibody. Equal amounts of analysed protein is shown by the expression of {alpha}-tubulin.

 
Furthermore, transfection of equal amounts of RSp53 or ASp53 coding plasmids into 174.2 p53/mdm-2 double knockout cells resulted in similar levels of p53 expression (Figure 2AGo). In contrast, transfection of equal amounts of RSp53 or ASp53 coding plasmids into murine K/O p53-null cells, which contain an intact endogenous mdm-2 gene, resulted in a significant difference in the level of expression of the two p53 protein variant forms (Figure 2BGo). It should be noted that RSp53 and ASp53 migrate on SDS–PAGE differently, due to the shortening of ASp53 by 9 amino acids at the p53 C-terminus (36). The reduced level of RSp53 expression, compared with that of ASp53, could be explained by the induction of the endogenous mdm-2 protein and the relative resistance of ASp53 to the mdm-2 directed degradation.

Although both of the p53 protein variants can equally bind mdm-2 (data not shown) and be translocated to the nucleus (data not shown), RSp53 is more efficiently directed to proteolytic degradation by mdm-2, than ASp53. Therefore, alteration of the extreme C' terminus of p53 (either by deletion of the RSp53 C' terminus, or by the acquisition of a unique and new C' terminus) accounts for the loss of sensitivity of ASp53 to mdm-2 dependent degradation.

RSp53 and ASp53 transcription activity is inhibited by mdm-2
The mdm-2 protein binds p53 at the N-terminal domain and thus inhibits p53-mediated transactivation (48,49). Since binding of mdm-2 to p53 is required for targeting p53 for degradation and since both of the p53 protein forms share the same N-terminus, we investigated the possibility that mdm-2 inhibits transactivation mediated by RSp53 and ASp53 variants. Using immuno-precipitation assays following transient transfections of either RSp53 or ASp53 together with mdm-2 coding plasmids, both p53 protein forms were shown to bind mdm-2 (data not shown). This is in agreement with the observation that only the p53 N-terminus is necessary for mdm-2 binding (48,49).

In order to examine whether these p53–mdm-2 protein interactions affect the transactivation capacity of RSp53 or ASp53, we transiently co-transfected RSp53 or ASp53 coding plasmids together with an empty vector or with an mdm-2 coding plasmid. As can be seen in Figure 3Go, addition of mdm-2 expression hardly affects the basal transcription level of the RGC and Waf-1 promoters, whereas a marked inhibition of both the RSp53- and ASp53-dependent transcription from both promoters following expression of mdm-2 was apparent. Following mdm-2 expression, the transactivation capacity of both RSp53 and ASp53 was reduced by 20–40%. The expression of mdm-2 reduced RSp53-dependent RGC transactivation by ~30%, whereas that of ASp53 was reduced by ~40%. The transactivation of the Waf-1 promoter by ASp53 was reduced by mdm-2 expression to a lesser extent (20%) than that by RSp53 (~30%). Therefore, it can be concluded that mdm-2 inhibits the transactivation capacities of both p53 protein variants to a similar extent.



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Fig. 3. Determination of the extent of mdm-2 mediated inhibition of p53-transciption activity. Luciferase assays were performed in 174.2 cell line. The cells were transiently co-transfected with a constant amount of luciferase reporter plasmid, carrying the luciferase gene under the control of either the RGC promoter (upper panel), or the Waf-1 promoter (lower panel). Equal amounts of expression plasmid, which contain either RSp53 or ASp53 cDNA, or an empty vector as a negative control, with or without the mdm-2 expression plasmid, were co-transfected with reporter plasmid, as indicated for each experiment. The amounts of RSp53 or ASp53 coding plasmids were constant in all of the transfections. The luciferase activity in each transfection was calculated according to the transfection efficiency, which was determined by ß-gal activity.

 
ASp53 inhibits RSp53-induced apoptosis through the induction of mdm-2
Previously we found that ASp53 was less efficient than RSp53 in inducing apoptosis in M1/2 myeloid cells. Furthermore, co-expression of RSp53 and ASp53 resulted in the inhibition of RSp53-induced apoptosis (42). This ASp53-dependent inhibition was specific for p53-dependent apoptosis, and was not evident in a p53-independent apoptotic pathway. Since the M1/2 cells contain an intact mdm-2 gene, we speculated that the ASp53-dependent inhibition of apoptosis might result from the induction of the mdm-2 protein expression by ASp53. This will in turn lead to the preferential degradation of RSp53 (due to the relative resistance of ASp53 to degradation), and thus will eventually attenuate the rate of apoptosis in these cells.

In order to examine this hypothesis we transiently transfected murine p53-null K/O cells with the RSp53 and ASp53 expressing plasmids. Twenty-eight hours following the transfection the apoptotic patterns of only the transfected cells was analysed by FACS according to the percentage of the sub-G1 fraction of the gated GFP-positive cells. The transfections were done in duplicate, and the results are presented as an average of the individual experiments. As can be seen in Figure 4AGo, a significant induction of apoptosis by RSp53 was observed. ASp53 was less efficient. Co-expression of both of p53 forms resulted in a lower level of apoptosis compared with that induced only by RSp53. This is in agreement with the previous observations in stable clones derived from the M1/2 myeloid cells (42), and further confirms that ASp53 expression inhibits the apoptosis induced by RSp53.



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Fig. 4. Evaluation of RSp53 and ASp53-dependent apoptosis. Either murine K/O p53-null cells (A), murine 174.2 mdm-2/p53 double knock-out cells (B) or human H1299 p53-null cells (C) were transiently transfected with RSp53, ASp53 or p19ARF coding plasmids, or with different combinations of the plasmids together with GFP-coding plasmid. An empty vector was used as a control. The cells were collected 48 (A, B) or 72 h (C) following transfection, stained with PI and the survival of only the transfected cells (GFP-positive cells) was determined by FACsort analysis. The apoptosis fraction is calculated as the percentage of the sub G1-fraction of each transfection and presented as the average of at least two independent transfections. (D) Western blot analysis for p19ARF. 1 µg (lane 1), 0.5 µg (lane 2) p19ARF expression plasmid or 1 µg empty vector (lane 3) were transiently transfected into H1299 p53-null cells. 72 h following transfection the cells were collected and analysed for p19ARF protein level using a monoclonal antibody directed against p19ARF.

 
In order to examine whether the preferential induction of mdm-2 is the cause of the inhibition of RSp53-dependent apoptosis observed above, we next co-transfected p53/mdm-2 double knockout 174.2 cells with RSp53 or ASp53 coding plasmids. As can be seen in Figure 4BGo, in these cells RSp53 was again more efficient in inducing apoptosis in comparison with ASp53. In contrast to that observed in the p53 null cells, only in the p53/mdm-2 double knock out cells did the co-expression of ASp53 with RSp53 not result in a decrease of the RSp53-induced apoptosis (Figure 4BGo). This may indicate that the inhibitory effect evident in the K/O and M1/2 cells is a result of the endogenous mdm-2 expression. Furthermore, although the total amount of transfected p53 DNA was equal in the last two transfections, addition of yet higher amounts of RSp53 coding plasmid resulted in an elevation of the apoptotic level, which was even higher than the level of apoptosis induced by the co-expression of RSp53 and ASp53. This observation may suggest that the two p53 protein forms share, at least in part, common mechanisms.

In order to further assess the role of mdm-2 in the inhibition of RSp53-induced apoptosis, we also transiently transfected human non-small cell lung carcinoma H1299, a p53-deficient cell line, with RSp53 and ASp53 coding plasmids. Seventy-two hours following transfection cells were collected and analysed for their apoptotic patterns (Figure 4CGo). Again, RSp53 induced higher levels of apoptosis compared with ASp53. As with K/O cells, in H1299 cells, co-expression of RSp53 and ASp53 resulted in an attenuation of the RSp53-induced apoptosis. Expression of higher amounts of RSp53 resulted in a significant enhancement of apoptosis compared with the level of apoptosis induced by the co-expression of RSp53 and ASp53 (although the final amount of transfected p53 coding plasmids is identical).

Another approach for determining the involvement of mdm-2 in the inhibitory effect mediated by ASp53 on RSp53 functions, could be through the abrogation with mdm-2 activity. Mdm-2 mediated degradation of p53 can be disrupted by the expression of the p19ARF protein [for review see (50)]. p19ARF is the product of the murine INK4A tumor suppressor gene, which binds both mdm-2 and p53 and thus abrogates the effects of mdm-2 on p53 protein (51,52). This is probably through the sequestering of mdm-2 into the nucleolus (53). In the following experiments we co-transfected the expression plasmids of p53 and p19ARF, which can be detected by western blot analysis (Figure 4DGo). As can be seen in Figure 4CGo, co-expression of p19ARF with p53 resulted in the enhancement of apoptosis induced by either RSp53 or ASp53. Most notably, p19ARF expression significantly altered the effect of ASp53 on RSp53-induced apoptosis. Most notably, in the presence of p19ARF, ASp53 synergizes with RSp53 in the induction of apoptosis, similar to the observed phenotype in 174.2 p53/mdm-2 double knockout cells. This suggests that the inhibitory effect of ASp53 expression on RSp53-induced apoptosis is mediated mostly through the mdm-2 protein, and that abolishment of mdm-2 activity, either by direct knocking out of the mdm-2 gene, or through interaction with p19ARF, eliminates the inhibitory effect mediated by ASp53.


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p53 is a short lived protein that was shown to be regulated by an mdm-2 dependent degradation pathway. Mdm-2 is a major component in the molecular mechanism which regulates the biological functions and protein levels of p53. Our present study adds an additional layer of complexity to the known p53/mdm-2 auto-regulatory loop and shows that in addition to the well established mechanisms by which p53 interacts with mdm-2 through its N-terminus, the C-terminus of p53 can also affect the p53/mdm-2 regulatory loop. We found that while ASp53 can selectively induce higher levels of the mdm-2 protein, compared with RSp53, it is relatively resistant to the mdm-2-directed proteolytic pathway. Furthermore, RSp53-induced apoptosis was shown to be inhibited by the addition of ASp53 expression, in an mdm-2 dependent manner.

Previously it was shown that p53 mutants which lack their extreme C' terminus can physically associate with mdm-2, but are resistant to mdm-2 directed degradation (32). In agreement with these results we show here that although the physiological variant of p53, ASp53, can bind mdm-2, and its transactivation capacity is inhibited by mdm-2, as is RSp53, it is resistant to mdm-2 dependent degradation. The molecular explanation for this selective resistance of ASp53 is still unknown. This could be the result of the inability of mdm-2 either to ubiquitinate ASp53, or to facilitate its export from the nucleus to the cytoplasm. Indeed, the ubiquitination of C-terminally deleted p53 proteins is markedly reduced (6).

The mechanisms which confer resistance of RSp53 to mdm-2 directed degradation following genotoxic stress were suggested to engage at least two pathways (for review see ref. 3). The first involves phosphorylation of several sites at the N' terminus of p53. The second includes interactions of either p53 or mdm-2 with other cellular proteins, such as c-abl or p19ARF. Since it is known that phosphorylation at certain sites of the p53 N-terminus domain are interconnected with modifications at the C' terminus, both of these possibilities might contribute to the enhanced stability of the ASp53 protein. Indeed, it was previously suggested that a third protein that binds the p53 C' terminus is required for the efficient targeting of p53 to degradation (32). Several cellular proteins were shown to bind the domain that is absent in the ASp53 protein. Most notably c-abl, which was shown to affect the proteolytic degradation of p53. Therefore, it could be suggested that p53 degradation is also a function of the protein conformation, which is largely regulated by the p53 C' terminus.

In this study, we have observed differences in the transcriptional activity and selectivity of the p53 protein variants. Although the two forms were shown to have the same DNA binding specificities (54), it is still unclear what is the basis for the preferential induction of mdm-2 expression by ASp53, and how the alteration of only the extreme C' terminus of p53 changes the patterns of transactivation. Since the most pronounced difference between these forms is in their ability to spontaneously bind specific DNA sequences (2426), it is still possible that the p53 variants differ in their binding affinities to the various p53 target sites. Another possibility is that cellular proteins which bind the p53 C' terminus might dictate the preferential transactivation patterns. Therefore, it is tempting to speculate that certain TFIIH associated factors, which were shown to bind the p53 C' terminus (16), could participate in this process. Interestingly, two classes of p53-responsive elements were shown to exist in the p21 promoter (55). The binding of p53 to these promoters was shown to be differentially regulated by the addition of PAb-421, which binds to the p53 extreme C' terminus. It is possible that the alterations in the p53 molecule following binding of PAb-421 are analogous to C-terminally modified p53 by post-translation modifications, proteolytic cleavages and alternative splicing. Therefore, it could be suggested that C-terminally altered p53 proteins may exhibit selective affinities for different classes of p53-responsive elements.

Moreover, p53 was shown to require p300 to transactivate mdm-2, while p300 co-transactivation is not required for other p53-inducible genes, such as Bax and p21 (56). p300 is known to acetylate the p53 C' terminus (10). It could be, therefore, that either the acetylation modification or the interaction with p300 itself might dictate the transactivation selectivities of the p53 protein variants.

Alternative splicing occurs also in the additional p53-family members, such as p73 and p63 (for review see ref. 57). Both of these p53 related proteins have at least three different isoforms that differ only in their C' terminus (p63{alpha}, -ß, -{gamma} and p73{alpha}, -ß, -{gamma}). These physiological variants possess altered biological activities, and were differentially distributed in various tissues (58,59). Moreover, the C-terminal domain of p73{alpha}, which is the most abundant isoform, was shown to be the regulatory domain of the protein, that regulates a variety of biological functions (60,61). Most notably, this domain regulates the p73 stability by the proteasome-dependent degradation of p73. These observations highlight the similarity in the processes that control the activity of p53 family members. This further strengthens the importance of the C' terminus of both p53 and p73 as regulatory units, which regulate protein degradation. It also substantiates the conclusion that p53 and p73 mRNA alternative splicing is a mechanism for controlling protein stability and activity. Interestingly, mdm-2 can bind p73 and inhibit its transactivation capability, but it does not direct p73 to degradation (for review see ref. 3).

p53 alternative splicing was shown to be conserved in rodent cells, and as observed in this study, it has a significant role in the regulation of p53 protein stability and activities. Although a human p53 alternatively spliced form has not yet been found, it is possible that alternative splicing reflects a general feature of p53 C' terminus modifications, which serves as a molecular regulatory mechanism to control the activity of these proteins. Indeed, a number of studies suggest that there may exist significant species-specific differences in the way p53 is modified and regulated (62,63) (for review see ref. 64). Since many types of post-translational modifications are known to occur on the p53 C' terminus, it could be suggested that human cells express certain p53 C-terminally modified proteins, which are the analogous forms of the murine ASp53.

In summary, it is possible that the relatively enhanced resistance of ASp53 to mdm-2 mediated protein degradation could serve as a mechanism for the constitutive stabilization of low levels of cellular p53, in the absence of cellular stress signals. Therefore, under normal growth conditions, the rate of the p53 mRNA alternative splicing might be important in determining the level and activity of the p53 protein.


    Notes
 
1 To whom correspondence should be addressedEmail: varda.rotter{at}weizmann.ac.il Back


    Acknowledgments
 
This work was supported in part by grants from Israel Cancer Association (ICA) and the Israeli–American Binational foundation (BSF). V.R. is the incumbent of the Norman and Helen Asher Professorial Chair in Cancer Research at the Weizmann Institute.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Giaccia,A.J. and Kastan,M.B. (1998) The complexity of p53 modulation: emerging patterns from divergent signals. Genes Dev., 12, 2973–2983.[Free Full Text]
  2. Sionov,R.V. and Haupt,Y. (1999) The cellular response to p53: the decision between life and death. Oncogene, 18, 6145–6157.[ISI][Medline]
  3. Ashcroft,M. and Vousden,K.H. (1999) Regulation of p53 stability. Oncogene, 18, 7637–7643.[ISI][Medline]
  4. Oren,M. (1999) Regulation of the p53 tumor suppressor protein. J. Biol. Chem., 274, 36031–36134.[Free Full Text]
  5. Fuchs,S.Y., Adler,V., Buschmann,T., Wu,X. and Ronai,Z. (1998) Mdm2 association with p53 targets its ubiquitination. Oncogene, 17, 2543–2547.[ISI][Medline]
  6. Honda,R., Tanaka,H. and Yasuda,H. (1997) Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett., 420, 25–27.[ISI][Medline]
  7. Shaulsky,G., Goldfinger,N., Ben-Ze'ev,A. and Rotter,V. (1990) Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis. Mol. Cell. Biol., 10, 6565–6577.[ISI][Medline]
  8. Stommel,J.M., Marchenko,N.D., Jimenez,G.S., Moll,U.M., Hope,T.J. and Wahl,G.M. (1999) A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J., 18, 1660–1672.[Abstract/Free Full Text]
  9. Meek,D.W. (1998) Multisite phosphorylation and the integration of stress signals at p53. Cell. Signal., 10, 159–166.[ISI][Medline]
  10. Gu,W. and Roeder,R.G. (1997) Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell, 90, 595–606.[ISI][Medline]
  11. Shaw,P., Freeamn,J., Bovey,R. and Iggo,R. (1996) Regulation of specific DNA binding by p53: evidence for a role for O-glycosylation and charged residues at the carboxy-terminus. Oncogene, 12, 921–930.[ISI][Medline]
  12. Malanga,M., Pleschke,J.M., Kleczkowska,H.E. and Althaus,F.R. (1998) Poly (ADP-ribose) binds to specific domains of p53 and alters its DNA binding functions. J. Biol. Chem., 273, 11839–11843.[Abstract/Free Full Text]
  13. Honda,R. and Yasuda,H. (1999) Association of p19 (ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53. EMBO J., 18, 22–27.[Abstract/Free Full Text]
  14. Okorokov,A.L., Ponchel,F. and Milner,J. (1997) Induced N- and C-terminal cleavage of p53: a core fragment of p53, generated by interaction with damaged DNA, promotes cleavage of the N-terminus of full length p53, whereas ssDNA induces C-terminal cleavage of p53. EMBO J., 16, 6008–6017.[Abstract/Free Full Text]
  15. Horikoshi,N., Usheva,A., Chen,J., Levine,J.A., Weinmann,R. and Shenk,T. (1995) Two domains of p53 interact with the TATA-binding protein and the Adenovirus 13S E1A protein disrupts the association, relieving p53-mediated transcriptional repression. Mol. Cell. Biol., 15, 227–234.[Abstract]
  16. Wang,X.W., Yeh,H., Schaeffer,L. et al. (1995) p53 modulation of TFIIH-associated nucleotide excision repair activity. Nature Genet., 10, 188–195.[ISI][Medline]
  17. Waterman,M.J.F., Stavridi,E.S., Waterman,J.L.F. and Halazonetis,T.D. (1998) ATM-dependent activation of p53 involves dephosphorylation and association with 14-4-3 proteins. Nature Genet., 19, 175–178.[ISI][Medline]
  18. Zhang,H., Somasundaram,K., Peng,Y., Tian,H., Zhang,H., Bi,D., Weber,B.L. and El-Deiry,W.S. (1998) BRCA1 physically associates with p53 and stimulates its transcriptional activity. Oncogene, 16, 1713–1721.[ISI][Medline]
  19. Delphin,C., Ronjat,M., Deloulme,J.C., Garin,G., Debussche,L., Higashimoto,Y., Sakaguchi,K. and Baudier,J. (1999) Calcium-dependent interaction of S100B with the C-terminal domain of the tumor suppressor p53. J. Biol. Chem., 274, 10539–10544.[Abstract/Free Full Text]
  20. Spillare,E.A., Robles,A.I., Wang,X.W., Shen,J.C., Yu,C.E., Schellenberg,G.D. and Harris,C.C. (1999) p53-mediated apoptosis is attenuated in Werner syndrome cells. Genes Dev., 13, 1355–1360.[Abstract/Free Full Text]
  21. Jayaraman,L., Murthy,K.G.K., Zhu,C., Curran,T., Xanthoudakis,S. and Prives,C. (1997) Identification of redox/repair protein Ref-1 as a potent activator of p53. Genes Dev., 11, 558–570.[Abstract]
  22. Li,H., Cao,Y., Berndt,M.C., Funder,J.W. and Liu,J.P. (1999) Molecular interactions between telomerase and the tumor suppressor protein p53 in vitro. Oncogene, 18, 6785–6794.[ISI][Medline]
  23. Rodriguez,M.S., Desterro,J.M., Lain,S., Midgley,C.A., Lane,D.P. and Hay,R.T. (1999) SUMO-1 modification activates the transcriptional response of p53. EMBO J., 18, 6455–6461.[Abstract/Free Full Text]
  24. Bayle,J.H., Elenbaas,B. and Levine,A.J. (1995) The carboxyl-terminal domain of the p53 protein regulates sequence-specific DNA binding through its nonspecific nucleic acid-binding activity. Proc. Natl Acad. Sci. USA, 92, 5729–5733.[Abstract]
  25. Wu,Y., Liu,Y., Lee,L., Miner,Z. and Kulesz-Martin,M. (1994) Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells. EMBO J., 13, 4823–4830.[Abstract]
  26. Wolkowicz,R., Elkind,N.B., Ronen,D. and Rotter,V. (1995) The DNA binding activity of wild type p53 is modulated by blocking its various antigenic epitopes. Oncogene, 10, 1167–1174.[ISI][Medline]
  27. Jayaraman,J. and Prives,C. (1995) Activation of p53 sequence-specific DNA binding by short single strands of DNA requires the p53 C-terminus. Cell, 81, 1021–1029.[ISI][Medline]
  28. Hupp,T.R., Meek,D.W., Midgley,C.A. and Lane,D.P. (1992) Regulation of the specific DNA binding function of p53. Cell, 71, 875–886.[ISI][Medline]
  29. Lee,S., Elenbaas,B., Levine,A. and Griffith,J. (1995) p53 and its 14 kDa C-terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches. Cell, 81, 1013–1020.[ISI][Medline]
  30. Reed,M., Woelker,B., Wang,P., Wang,Y., Anderson,M.E. and Tegtmeyer,P. (1995) The C-terminal domain of p53 recognizes DNA damaged by ionizing radiation. Proc. Natl Acad. Sci. USA, 92, 9455–9459.[Abstract]
  31. Crook,T., Ludwig,R.L., Marston,N.J., Willkomm,D. and Vousden,K.H. (1996) Sensitivity of p53 lysine mutants to ubiquitin-directed degradation targeted by human papillomavirus E6. Virology, 217, 285–292.[ISI][Medline]
  32. Kubbutat,M.H.G., Ludwig,R.L., Ashcroft,M. and Vousden,K.H. (1998) Regulation of Mdm2-directed degradation by the C terminus of p53. Mol. Cell. Biol., 18, 5690–5698.[Abstract/Free Full Text]
  33. Gostissa,M., Hengstermann,A., Fogal,V., Sandy,P., Schwarz,S.E., Scheffner,M. and Del Sal,G. (1999) Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1. EMBO J., 18, 6462–6471.[Abstract/Free Full Text]
  34. Sionov,R.V., Moallem,E., Berger,M., Kazaz,A., Gerlitz,O., Ben-Neriah,Y., Oren,M. and Haupt,Y. (1999) c-Abl neutralizes the inhibitory effect of Mdm2 on p53. J. Biol. Chem., 274, 8371–8374.[Abstract/Free Full Text]
  35. Nie,Y., Li,H.H., Bula,C.M. and Liu,X. (2000) Stimulation of p53 DNA binding by c-Abl requires the p53 C-terminus and tetramerization. Mol. Cell. Biol., 20, 741–748.[Abstract/Free Full Text]
  36. Arai,N., Nomura,D., Yokota,K., Wolf,D., Brill,E., Shohat,O. and Rotter,V. (1986) Immunologically distinct p53 molecules generated by alternative splicing. Mol. Cell. Biol., 6, 3232–3239.[ISI][Medline]
  37. Kulesz-Martin,M.F., Lisafeld,B., Huang,H., Kisiel,N.D. and Lee,L. (1994) Endogenous p53 protein generated from wild-type alternatively spliced p53 RNA in mouse epidermal cells. Mol. Cell. Biol., 14, 1698–1708.[Abstract]
  38. Will,K., Warnecke,G., Bergmann,S. and Deppert,W. (1995) Species- and tissue-specific expression of the C-terminal alternatively spliced form of the tumor suppressor p53. Nucleic Acids Res., 23, 4023–4028.[Abstract]
  39. Magnusson,K.P., Satalino,R., Qian,W., Klein,G. and Wiman,K.G. (1998) Is conversion of solid into more anoxic ascites tumors associated with p53 inactivation? Oncogene, 17, 2333–2337.[ISI][Medline]
  40. Wu,L., Bayle,H., Elenbaas,B., Pavletich,N.P. and Levine,A.J. (1995) Alternatively spliced forms in the carboxy-terminal domain of the p53 protein regulate its ablity to promote annealing of complementary single strands of nucleic acids. Mol. Cell. Biol., 15, 497–504.[Abstract]
  41. Almog,N., Li,R., Peled,A., Schwartz,D., Wolkowicz,R., Goldfinger,N., Pei,H. and Rotter,V. (1997) The murine C'-terminally alternatively spliced form of p53 induces attenuated apoptosis in myeloid cells. Mol. Cell. Biol., 17, 713–722.[Abstract]
  42. Almog,N., Goldfinger,N. and Rotter,V. (2000) p53-dependent apoptosis is regulated by a C-terminally alternatively spliced form of murine p53. Oncogene, 19, 3395–403.[ISI][Medline]
  43. McMasters,K.M., Montes de Oca Luna,R., Pena,J.R. and Lozano,G. (1996) mdm2 deletion does not alter growth characteristics of p53-deficient embryo fibroblasts. Oncogene, 13, 1731–1736.[ISI][Medline]
  44. Wu,Y., Huang,H., Miner,Z. and Kulesz-Martin,M. (1997) Activities and response to DNA damage of latent and active sequence-specific DNA binding forms of mouse p53. Proc. Natl Acad. Sci. USA, 94, 8982–8987.[Abstract/Free Full Text]
  45. Barak,Y., Gottlieb,E., Juvengershon,T. and Oren,M. (1994) Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential. Gene Dev., 8, 1739–1749.[Abstract]
  46. Haupt,Y., Maya,R., Kazak,A. and Oren,M. (1997) Mdm2 promotes the rapid degradation of p53. Nature, 387, 296–299.[ISI][Medline]
  47. Kubbutat,M.H.G., Jones,S.N. and Vousden,K.H. (1997) Regulation of p53 stability by Mdm2. Nature, 387, 299–303.[ISI][Medline]
  48. Oliner,J.D., Pietenpol,J.A., Thiagalingam,S., Gyuris,J., Kinzler,K.W. and Vogelstein,B. (1993) Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53. Nature, 362, 857–860.[ISI][Medline]
  49. Momand,J., Zambetti,G.P., Olson,D.C., George,D. and Levine,A.J. (1992) The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell, 69, 1237–1245.[ISI][Medline]
  50. Chin,L., Pomerantz,J. and DePinho,R.A. (1998) The INK4a/ARF tumor suppressor: one gene–two products–two pathways. Trends Biochem. Sci., 23, 291–296.[ISI][Medline]
  51. Kamijo,T., Weber,J.D., Zambetti,G., Zindy,F., Roussel,M.F. and Sherr,C.J. (1998) Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc. Natl Acad. Sci. USA, 95, 8292–8297.[Abstract/Free Full Text]
  52. Stott,F., Bates,S., James,M., McConnell,B., Starborg,M., Brookes,S., Palmero,I., Ryan,K., Hara E., Vousden,K. and Peters G. (1998) The alternative product from the human CDKN2A locus, p14 (ARF), participates in a regulatory feedback loop with p53 and MDM2. EMBO J., 1, 5001–5014.
  53. Weber,J.D., Taylor,L.J., Roussel,M.F., Sherr,C.J. and Bar-Sagi,D. (1999) Nucleolar Arf sequesters Mdm2 and activates p53. Nature Cell. Biol., 1, 20–26.[ISI][Medline]
  54. Miner,Z. and Kulesz-Martin,M. (1997) DNA binding specificity of proteins derived from alternatively spliced mouse p53 mRNAs. Nucleic Acids Res., 25, 1319–1326.[Abstract/Free Full Text]
  55. Resnick-Silverman,L., St Clair,S., Maurer,M., Zhao,K. and Manfredi,J.J. (1998) Identification of a novel class of genomic DNA-binding sites suggests a mechanism for selectivity in target gene activation by the tumor suppressor protein p53. Genes Dev., 12, 2102–2107.[Abstract/Free Full Text]
  56. Thomas,A. and White,E. (1998) Suppression of the p300-dependent mdm2 negative-feedback loop induces the p53 apoptotic function. Genes Dev., 12, 1975–1985.[Abstract/Free Full Text]
  57. Kaelin,W.G.Jr (1999) The p53 gene family. Oncogene, 18, 7701–7705.[ISI][Medline]
  58. Yang,A., Kaghad,M., Wang,Y., Gillett,E., Fleming,M.D., Dotsch,V., Andrews,N.C., Caput,D. and McKeon,F. (1998) p63, a p53 homolog at 3q27-29, encodes multiple products with transactivating, death-inducing and dominant-negative activities. Mol. Cell, 2, 305–316.[ISI][Medline]
  59. De Laurenzi,V., Costanzo,A., Barcaroli,D., Terrinoni,A., Falco,M., Annicchiarico-Petruzzelli,M., Levrero,M. and Melino,G. (1998) Two new p73 splice variants, {gamma} and {delta}, with different transcriptional activity. J. Exp. Med., 188, 1763–1768.[Abstract/Free Full Text]
  60. Ozaki,T., Naka,M., Takada,N., Tada,M., Sakiyama,S. and Nakagawara,A. (1999) 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 cells. Cancer Res., 59, 5902–5907.[Abstract/Free Full Text]
  61. Lee,C. and Thangue,N.B. (1999) Promoter specificity and stability control of the p53-related protein p73. Oncogene, 18, 4171–4181.[ISI][Medline]
  62. Laverdiere,M., Beaudoin,J. and Lavigueur,A. (2000) Species-specific regulation of alternative splicing in the C-terminal region of the p53 tumor suppressor gene. Nucleic Acids Res., 28, 1489–1497.[Abstract/Free Full Text]
  63. Jardine,L.J., Milne,D.M., Dumaz,N. and Meek,D.W. (1999) Phosphorylation of murine p53, but not human p53, by MAP kinase in vitro and in cultured cells highlights species-dependent variation in post-translational modification. Oncogene, 18, 7602–7607.[ISI][Medline]
  64. Meek,D.W. (1999) Mechanisms of switching on p53: a role for covalent modification? Oncogene, 18, 7666–7675.[ISI][Medline]
Received September 22, 2000; revised January 16, 2001; accepted January 24, 2001.





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