response pathways and tumorigenesis
Bruins, W.
Citation
Bruins, W. (2007, October 24). The role of p53.S389 phosphorylation in DNA damage response pathways and tumorigenesis. Department Toxicogenetics, Medicine / Leiden University Medical Center (LUMC), Leiden University.
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Chapter 6
Phosphorylation of p53 at serine 389 is compound-
specific and possibly initiated by stalled RNA
polymerases
Wendy Bruins Edwin Zwart Harry van Steeg Annemieke de Vries
Abstract
Post-translational modifications, and in particular phosphorylation, have an important role in the activation of p53 and in the regulation of its cellular responses. Here we investigated the role of p53.S389 phosphorylation in particular in different biological processes.
With the use of different genotoxic compounds giving rise to DNA damages that are substrate to NER and/or BER we could show that phosphorylation of p53.S389 is not only triggered by compounds that lead to DNA damage that is substrate to NER. Although phosphorylation of p53.S389 seems not to be directly related to compounds inducing DNA damage that is substrate to NER, the relative p53.S389 phosphorylation levels are, however, most abundant after exposure to UV. Furthermore, this phosphorylation event was found not to be specifically related to a p53-dependent apoptotic or cell-cycle arrest response in primary cultured MEFs.
We have previously shown that p53.S389A mutant mice are more susceptible for the induction of skin tumors after UV exposure and urinary bladder tumors formation after 2-AAF exposure.
Since both UV and 2-AAF are inducing DNA damage that is substrate to NER, we further investigated the role of p53.S389 phosphorylation and its possible relationship to NER.
Our findings suggest that the induction of p53.S389 phosphorylation during the p53 response may be linked to DNA lesions inducing blockage of the elongation of RNA-polymerase. We cannot, although more unlikely, rule out the possibility that p53.S389 phosphorylation results from blocked DNA replication as well. Activation of p53 in response to DNA damage is a complex multiple signaling event of which phosphorylation of p53.S389 is needed for some, but certainly not all parts.
Introduction
Upon DNA-damage, a variety of cellular responses are initiated. For instance, the p53 protein is stabilized and activated, resulting in the induction of cellular responses like apoptosis or cell cycle arrest by transcriptional activation of a large group of its target genes (reviewed in [1-3]).
These cellular responses prevent the survival of cells with altered genomes and, as such, protect the cell from becoming cancerous.
A well-known mechanism to activate p53 is post-translational modification by means of phosphorylation. P53 can be phosphorylated in vitro by several different kinases, and phosphorylation has been shown to occur at both its N- and C-terminus. It is hypothesized that specific types of DNA damages or the level of DNA damage directs the specific (cascade of) phosphorylation events, and as a result, the way the cell responds (reviewed in [3-9]). Still, it is largely unknown how p53 decides which cellular defense pathway it activates upon stress.
Initially, it has been suggested that low p53 protein levels lead to cell cycle arrest and that this process is driven by p53-downstream genes having high affinity promoters. In line with this it was assumed that apoptosis takes place under conditions of high p53 protein levels and that responding genes carry low p53 affinity promoters. However, this latter hypothesis dealing with apoptosis was rejected, since for example the pro-apoptotic gene PUMA appears to have a high affinity promoter [10-12] (also reviewed in [2]).
Another broadly acknowledged hypothesis is that modulation of the p53-dependent apoptotic response is dependent on auxiliary proteins. Some of these p53-associated factors have been shown to have a specific and selective role in enabling the expression of apoptotic target genes, for example the JMY protein or members of the ASPP-family can favor the binding of p53 protein with promoters of apoptotic genes [2;13]. Finally, Oren postulated the decision made
by p53 between apoptosis and cell cycle arrest is more dependent on the cellular context, which is the balance between intra- and extracellular signaling events [14]. Overall, the specific cell type, the type and intensity of DNA damage and the extent to which p53 interacts with co-activators or regulators seem to be very important in directing the final cellular response.
Recently, however, phosphorylation of serine 46 [15] as well as acetylation of lysine 120 [16]
were identified as important post-translational modifications of p53 in regulation of the p53- dependent apoptotic response.
When the cell responds to DNA damage through the initiation of cell cycle arrest, it is presumably to allow the damage to be repaired. Depending on the specific type of DNA damage, one of several known DNA repair pathways will become actived to repair the damage. The best studied repair mechanism is the nucleotide excision repair pathway (NER), which is involved in the removal of a broad range of DNA lesions [reviewed in [17]]. This repair system recognizes lesions disturbing the double strand DNA helix, like bulky adducts or cross-links [reviewed in [18;19]]. NER comprises two sub-pathways: global genome repair (GG-NER) and transcription coupled repair (TC-NER). GG-NER removes lesions from the global genome and from the non-transcribed strand of active genes. TC-NER is initiated by stalled RNA-polymerase II, removing these lesions from the transcribed strand of active genes [20;21]. Interestingly, DNA damage in actively transcribed regions is repaired much more efficiently compared to DNA damage in the genome overall (reviewed in [22;23]). Evidence for a potential direct involvement of p53 in NER came from the observation that human cells lacking p53 have an impaired capacity to repair UV-induced lesions [24;25]. Looking in more detail it appeared that p53 is required for efficient GG-NER and not for TC-NER [26;27]. In another study, however, it was found that p53 has a role in TC-NER as well [25;28], indicating that the interactions between p53 and DNA repair are complex and that further studies are urgently needed.
Another eminent repair system is base excision repair (BER), involved in repairing DNA damage induced by endogenous cellular metabolism (methylation, deamination, ROS and byproducts of hydrolysis) and maintaining genome integrity by correcting DNA base modifications [29]. It has been reported that p53 can stimulate BER, and specifically a direct interaction of p53 with the endonuclease APE or with the DNA-polymerase-β was suggested [29-31].
It has been shown before that induction of p53 can be triggered when transcription elongation is blocked [32-34]. Interestingly, Ljungman et al. described that post-translational modification of Ser15 and Lys382 are important in this process [35]. These post-translational modifications seem to be specifically triggered by blockage of elongating RNA polymerase II complexes and not directly by inhibiting the RNA polymerase itself [35]. Moreover, it was found that prolonged inhibition of mRNA synthesis can result in apoptosis [35]. Therefore, it is crucial to the cell to initiate a quick response to DNA damage in transcribed strands. The exact mechanism on how accumulation of p53 occurs in this context is, however, not entirely understood [36].
In addition, how p53-dependent cellular processes are related to DNA repair is also largely unknown.
Mouse models with specific modifications in p53 or individual NER genes might be suitable tools to study these complex interactions. In this context the p53.S389A mouse [37] developed by us could be an attractive model. We recently showed that this mouse mutant is sensitive to UV and 2-AAF exposure. These agents both induce DNA lesions that are substrate to NER [37;38]. In contrast, it was found that p53.S389A mice behave like wild-type mice when they
are exposed to compounds that do not give DNA lesions that are substrate to NER, e.g., upon exposure to gamma irradiation. These findings suggest that phosphorylation at Ser389 has a compound- or DNA repair pathway-dependent component.
Here, we tested the hypothesis that a specific type of DNA damage directs the cellular response by phosphorylation of specific p53 residues. For this, we characterized the effects in terms of Ser389 phosphorylation of several agents causing a type of DNA damage mainly repaired by either NER or BER.
Finally, we analyzed whether phosphorylation of p53.S389 has a role in activation of p53 in the case that transcription elongation becomes blocked owing to accumulated DNA lesions in actively transcribed genes. To address this, MEFs were used isolated from mice with various defects in NER, and these cells were subsequently exposed to UV after which the phosphorylation status of p53 at residue Ser389 was determined.
All together, our results indicate that TC-NER and p53 phosphorylation at Ser389 are closely related, and as such, reveals new evidence for a direct link between p53 and NER.
Materials and Methods
Western Blot analysis
Primary MEFs (wild-type, p53.S389A, Xpa-/- , Xpc-/- and Csb-/-) prior to passage 5 were expanded in culture flasks (Greiner) and plated at 0.7*106 cells per 10 cm plate (Greiner). 24 hours later, cells were washed with PBS and treated with several DNA damaging agents (20 J/m2 UV-C, 30 µM N-Acetoxy-AAF dissolved in DMSO, 1 mM MNU dissolved in Sorensenbuffer pH 6.0 [11.88 g/l Na2HPO4.2H2O, 9.08 g/l KH2PO4], 7.5 µg/ml paraquat dissolved in MilliQ, or 2 µg/ml doxorubicin dissolved in MilliQ). MEFs were treated during the whole experiment except for UV-C, which was one exposure at t=0. The doses used were based on earlier exposure studies [37;39]. At several time points after the treatment, MEFs were rinsed with PBS and each sample was collected in 50 µl ice cold lysis buffer ([100 mM Tris-HCl (pH 8.0),100 mM NaCl, 1% Triton X-100, 10% Glycerol], supplemented with complete mini protease inhibitor cocktail tablets (Roche) and phosphatase inhibitor cocktail 1 and 2 (Sigma)). Cells were lysed by rotating them at 4°C for 30 min., followed by 15 min of centrifugation (20,000xg) at 4ºC to remove cellular debris. Total protein concentrations were determined using the BCA Protein Assay Kit (Pierce).
To detect p53, immunoprecipitation on 100 µg total protein was applied as described previously [37]. Membranes were incubated for at least 12 hours at 4ºC with either anti-p53 mouse monoclonal antibody (Ab-1; Oncogene Research Products) or anti-phospho-p53 rabbit polyclonal antibodies (Ser392 and Ser15; Cell Signaling). Incubation for 1 hour at RT with horseradish peroxidase (HRP) linked anti-actin affinity-purified goat polyclonal antibody (I-19- HRP; Santa Cruz) was performed on the membrane with total cell extract. Primary antibodies were detected by incubating for 1 hour at RT with HRP-linked sheep-anti mouse IgG or HRP-linked donkey-anti rabbit IgG (Amersham Pharmacia Biotech), and staining was done using ECL-plus reagent (Amersham Pharmacia Biotech). Membranes were scanned using a PhosphorImager/Storm 860 (Molecular Dynamics) and ratios were determined by TotallabTM version 2.00 by using the actin protein as a loading control (Nonlinear Dynamics).
Analysis of apoptosis and cell cycle arrest in MEFs
Primary MEFs (wild-type, p53.S389A, p53-/-) were prepared and cultured as described before
[40]. Experiments were performed with early passage MEFs (prior to passage 5). For analysis of apoptosis, MEFs were expanded, and plated onto 24-well plates (3*104 cells per well) (Greiner). After 24 hours the cells were washed with PBS and exposed to the different DNA damaging agents at the indicated concentrations. For the cell death detection enzyme-linked immunosorbent assay ELISA-kit (Roche Molecular Biochemicals), adherent cells were lysed 16 hours after the treatment. Apoptotic levels in each well (based on the enrichment of mono- and oligonucleosomes in the cytoplasm of the apoptotic cells) were subsequently determined according to the manufacturer’s instructions.
For cell cycle analysis, MEFs (wild-type, p53.S389A, p53-/-) were synchronized in G0 by growing them to confluence followed by serum starvation. Cells were subsequently trypsinized and replated at 1*106 cells per 10 cm plate (Greiner) in the presence of 10% serum to allow re-entering into the cell cycle. After 6 hours, cells were exposed to the same panel of DNA damaging compounds as described above for analysis of apoptosis, and MEFs were collected, prepared, and Fluorescent Activated Cell Sorting (FACS) analyses were performed as described earlier [41]. After FACS analysis, fractions of cells in each phase of the cell cycle were quantified using CellQuest software (Becton Dickinson).
Results
P53 protein levels and phosphorylation of p53.S389 after exposure to various DNA damaging agents
First, we addressed the question whether p53.S389 phosphorylation occurs preferentially upon exposure to compounds that lead to DNA damage that is substrate to NER. To answer this question we started a series of experiments in which we exposed primary MEFs to different genotoxic compounds that give rise to lesions that are substrate to NER and/or other DNA repair pathways, like UV and 2-AAF (NER) or MNU and paraquat (BER). Next, total p53 protein levels and the portion of p53 that became phosphorylated at Ser389 were determined by Western blot analysis. Results are shown in Figure 1A and 1B. Overall levels of total p53 protein and Ser389 phosphorylated p53 significantly increased over time after exposure to UV or 2-AAF, whereas exposure to MNU resulted in lower increases of protein levels over time.
Interestingly, exposure to paraquat did not result in increased p53 protein levels at all, and consequently did not result in the accumulation of the Ser389 phosphorylated version of the protein. Apparently, paraquat-induced (oxidative?) DNA damage does not activate p53 even at higher doses of paraquat tested (upto 15 µg/ml, results not shown).
To more quantitatively compare p53 protein levels, we measured the absolute intensities of the protein using actin as a loading control. Figure 1B shows the induction of p53 protein levels and p53.S389 phosphorylation levels in time both corrected for loading differences. After exposure to UV and 2-AAF both compounds leading to DNA damage that is substrate to NER, an about 25-fold increase in total p53 levels was observed compared to untreated MEFs. A 22-fold induction of the Ser389 phosphorylated form was found after exposure to UV, whereas for 2- AAF this value was only 11-fold. For the more ’BER specific’ compounds, only MNU exposure resulted in a 14-fold increase of total p53 levels, and a 13-fold increase in levels of Ser389 phosphorylated p53. In conclusion, induction of total p53 protein levels is higher in MEFs exposed to UV or 2-AAF as compared to MNU, with relative Ser389 phosphorylation protein levels being most abundant after exposure to UV. Thus, phosphorylation of p53.S389 seems not to be solely restricted to compounds that lead to DNA damage that is substrate to NER.
Cellular responses
An important cellular response to DNA damage triggered by p53 is apoptosis [42]. Besides apoptosis the cell can also respond to the DNA damage through cell cycle arrest, to allow cells to repair their DNA damage. For some DNA damaging compounds it is well known what the ultimate cellular response is in MEFs. Gamma irradiation, for instance, does not induce apoptosis, but exclusively induces a p53-dependent G1-cell cycle arrest [43;44]. Exposure to UV, in contrast, gives dependent on the dose rise to a p53-dependent apoptotic response or a p53-independent G1-cell cycle arrest [37;39]. The occurrence of specific post-translational events on p53 is thought to play an important role in this decision. Here, we studied whether phosphorylation at serine 389 of p53 plays a role in cells to go either into apoptosis, and/or into a cell cycle arrest.
Apoptosis
It had been shown before [43] that MEFs treated with gamma irradiation, doxorubicin or etoposide do not go into apoptosis. Furthermore, in Chapter 2 of this thesis, we have shown that exposure of MEFs to UV induces a p53-dependent apoptotic response, which is decreased in the p53.S389A mutant MEFs (Figure 2A, left panel). Since we were interested in the potential role of phosphorylation of p53.S389 in directing cells into a p53-dependent apoptotic response we tested the response of MEFs exposed to 2-AAF and MNU (both induce phosphorylation of p53.S389) and paraquat which hardly showed any induction of p53.S389 phosphorylation.
To our surprise, only exposure to 2-AAF led to an apoptotic response, and moreover this response appeared to be p53-independent (Figure 2A, right panel). No detectable levels of apoptotic cells could be observed after exposure to MNU or paraquat. Apparently, phosphorylation of p53.S389 is not the only determining factor directing cells into apoptosis. Induction of apoptosis seems, at least to some extent, to be dependent on the intrinsic features of compounds as well.
2-AAF
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Figure 1 - Western blot analysis of wild-type MEFs exposed to different DNA damaging agents
A) Phosphorylation of p53.S389 and total p53 protein levels in wild-type MEFs after exposure to 20 J/m2 UV-C radiation, 6 µM 2-AAF, 1mM MNU or 7.5 µg/ml paraquat. Protein extracts were prepared from MEFs at different time points as indicated after the treatment and phosphorylation of Ser389 and total p53 protein levels were visualized by Western blotting after immunoprecipitation. Total protein extract was loaded onto an additional Western blot, which was incubated with actin antibody, to control for loading differences.
B) Semi-quantitative analysis of the Western blot performed by scanning the protein bands and determining the fold induction levels of both p53 protein levels and phosphorylation levels of Ser389 using actin protein levels as a loading control and the untreated samples as basal level (=1). Black; wild-type, grey; p53.S389A.
Cell cycle arrest analysis
Effects of DNA damaging agents on cell cycle arrests were determined using FACS analyses. A representation of these analyses is shown in Figure 2B. Untreated MEF samples representing all genotypes show a normal cell cycle distribution, indicating that inactivation of Ser389 or complete loss of p53 does not affect normal cell cycling. However, for example exposure to UV induced a clear G1- and G2-cell cycle arrest in wild-type MEFs and hardly any cells went into
Wild-type
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Absorbance405-490nm Wild-type
p53.S389A p53-/-
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UV Relativeapoptoticresponse (16hrsafterUV)
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Figure 2 - Apoptosis and cell cycle arrest in MEFs exposed to different DNA damaging agents
A) Wild-type, p53.S389A and p53-/- MEFs were exposed to 20 J/m2 UV-C radiation, 6 µM 2-AAF, 1mM MNU or 7.5 µg/ml paraquat.
The apoptotic responses were measured 16 hours later with a cell death ELISA detection assay. The relative apoptotic responses in the UV-irradiated samples, normalized to the apoptotic responses in untreated cultures from the same MEF cell line, is depicted in the left panel [37]. The apoptotic responses after 2-AAF, MNU and paraquat are depicted in the right panel (i.e., higher absorbance level indicates a higher apoptotic response). For each data point, the mean of at least three independent measurements is shown.
B) Wild-type, p53.S389A and p53-/- MEFs were synchronized and exposed to 20 J/m2 UV-C radiation, 6 µM 2-AAF, 1mM MNU or 7.5 µg/ml paraquat. BrdU was added during the last 4 hours of the 18 hours treatment period to label cells in the S-phase. Cell cycle profile was determined by FACS analysis. Cells were sorted for BrdU incorporation and DNA content (PI). Representative examples of the FACS analysis pictures of treated MEFs are shown. The G1-, S- and G2/M- phase are indicated in the upper left example.
the S-phase. Comparable profiles were also observed in p53.S389A and p53-/- MEFs, indicating that the UV-induced G1- and G2-cell cycle arrest is p53-independent. Furthermore, exposure to 2-AAF, MNU or paraquat did not result in different cell cycle profiles between wild-type, p53.S389A and p53-/-.
In conclusion, we showed that phosphorylation of p53.S389 does not have a role in directing primary MEFs into either apoptosis or cell cycle arrest events. Again, the cellular response appears to be more dependent on the compound than on p53.S389 phosphorylation. This might indicate that MEFs are no suitable cells to study these (p53 dependent) cellular processes.
Repair defects and phosphorylation of p53.S389
So far, our results do not show a direct relationship between phosphorylation of p53.S389 and a final p53-dependent cellular response. In addition, no conclusive correlation between DNA damage that is substrate to NER and phosphorylation of p53.S389 could be demonstrated, since also other compounds give rise to this phosphorylation event. Clearly, however, DNA damages that are substrate to NER, especially those induced by UV do result in absolute stronger levels of serine 389 phosphorylation of p53 compared to others (Figure 1).
In repair-proficient mammalian cells, UV-induced DNA lesions were shown to be exclusively repaired by NER [45]. We were, therefore, interested if deletion of p53 or inactivation of p53.S389 phosphorylation has an influence on NER activity. We, therefore, measured the NER activity in cultured cells by the UV-induced unscheduled DNA synthesis (UDS) assay, which is a measure for global genome repair (GG-NER). No differences in UDS activity between the different genotypes were observed (data not shown). Based on these results we hypothesized that persistent DNA damage in actively transcribed DNA, resulting in a blockage of transcription, might well be the initiating stimulus of phosphorylation of p53.S389. In line with this thinking is the observation [35] that a correlation exists between blockage of transcription elongation and phosphorylation of p53.S15.
We, therefore, specifically focused on defects in NER. MEFs were isolated from mice that are repair proficient (wild-type), GG-NER deficient (Xpc-/-), TC-NER deficient (Csb-/-) or deficient in both GG-NER and TC-NER (Xpa-/-) (see also Figure 3A). These MEFs were exposed to 4 J/m2 and 12 J/m2 UV radiation and p53 protein levels were identified in time (Figure 3B).
Interestingly, the Xpa-/- and Csb-/- MEFs, both lacking TC-NER, clearly showed an earlier induction of p53.S389 phosphorylation and total p53 protein levels compared to wild-type and Xpc-/- MEFs. This was already apparent after 4 hours in MEFs exposed at 4 J/m2 of UV radiation.
In contrast, in wild-type and Xpc-/- MEFs hardly any induction of p53 protein levels was found at this low dose, not even at later time points. After exposure to 12 J/m2 UV radiation all MEFs, irrespective of their genotype, showed an induction of both p53.S389 phosphorylation and total p53 protein levels. Strongest inductions were again detected in Xpa-/- and Csb-/- MEFs as compared to wild-type and Xpc-/- MEFs.
In conclusion, it seems that a defect in TC-NER results in a rapid and higher induction of total p53 protein levels, which is accompanied by comparable phosphorylated p53.S389 levels. In cells lacking only GG-NER, phosphorylation of p53.S389 is comparable to levels found in wild-type cells. To find out whether these observations are restricted to compounds that lead to DNA damage that is substrate to NER we exposed the different NER-deficient MEFs to the compound MNU, introducing DNA damages which are no substrate to NER, and we again analyzed total p53 protein levels and induction of p53.S389 phosphorylation. It appeared that
0 4 6 8 16 24 4 8 16 24 Wild-type
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Adapted and modified from: J.H.J. Hoeijmakers, Nature, 2001
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Figure 3 - Western blot analysis of NER-deficient MEFs exposed to UV radiation
A) Figure presenting a summary of the different defects in the NER sub-pathways of the MEFs used for this analysis.
Furthermore, an overview of the NER-system in mammalian cells is shown, representing the different steps and proteins involved; damage recognition, DNA unwinding, incision of the DNA strand, excision of damaged DNA and de novo synthesis of DNA.
B) Phosphorylation of p53.S389 and total p53 protein levels in MEFs with complementary defects in the NER sub-pathways after exposure to UV at the indicated doses.
Protein extracts were prepared from MEFs at different time points as indicated. After exposure to 4 or 12 J/m2 UV-C, phosphorylation levels of p53.Ser389 and total p53 protein levels were visualized by Western blotting and immunoprecipitation. Total extracts were loaded onto an additional Western blot, which was incubated with actin antibody to correct for loading differences.
C) Phosphorylation of p53.S389 and total p53 protein levels in MEFs with complementary defects in the NER sub-pathways at different time points upon 1mM MNU treatment.
the total p53 protein levels and induction of p53.S389 phosphorylation are highly comparable in wild-type, Xpa-/- , Xpc-/- and Csb-/- MEFs (Figure 3C).
Stalled RNA polymerases and p53.S389 phosphorylation
Defects in the repair of UV-induced DNA damage in actively transcribed DNA seems to initiate p53.S389 phosphorylation, pointing towards a link between stalled RNA polymerases and p53.S389 phosphorylation.
To investigate whether it is the specific type of DNA damage or the stalled polymerase itself that triggers the p53.S389 phosphorylation, we exposed the NER-deficient MEFs to doxorubicin.
Doxorubicin has an inhibitory effect on RNA polymerase II prior to or during elongation [46- 49]. From Figure 4 it is clear that both p53.S389 phosphorylation and p53 protein accumulation occurred upon doxorubicin exposure, underlining the hypothesis that stalled RNA polymerases (or blocked DNA replication) trigger p53 phosphorylation. No differences in p53 activation could be detected between the different NER-deficient and wild-type cells upon doxorubicin exposure. This indicates that the differences in the NER-deficient cells with regards to p53.S389 phosphorylation are due to the accumulation of stalled polymerases resulting from persistent DNA damage in actively transcribed DNA.
Discussion
P53 has, depending on the cellular context, an important role in DNA damage response pathways, inducing either cell cycle arrest or apoptosis [1]. P53 needs to be activated to adequately initiate these cellular responses. Post-translational modifications, like phosphorylation of the protein, are involved in the regulation of the stability and activity of the p53 protein. Several research groups [50;51] have studied these regulatory processes in vitro. In addition to these in vitro studies, also some mutant mouse models were made to investigate the contribution of different phosphorylation sites to the p53-dependent cellular responses after induction to DNA damage.
Cells derived from mouse models containing germ-line mutations at serine 18, serine 23 or at both serines, disabling phosphorylation of the residues, have shown to be compromised in their apoptotic responses [52-55]. The cell cycle arrest responses, however, were either normal or decreased depending on the residue mutated and the DNA damaging agent used. We focused on the post-translational modification at p53.S389, a phosphorylation event (specifically) described to occur after exposure to UV radiation, but not after gamma irradiation [56;57].
We have recently shown that the p53.S389A mutation results in increased tumor-susceptibility of mice after exposure to UV and, furthermore, to a decreased apoptotic response in MEFs [37]. Also exposure to 2-AAF resulted in an increased tumor incidence in urinary bladders of p53.S389A mice. In contrast, tumor and cell cycle arrest responses upon exposure to
Time (hours)
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XPA
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p53.S389p Actin p53
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Figure 4 - Western blot analysis of NER-deficient MEFs exposed to doxorubicin
Phosphorylation of p53.S389 and total p53 protein levels in MEFs with complementary defects in the NER sub-pathways at different time points upon doxorubicin treatment
gamma irradiation were indistinguishable from wild-type cells. This clearly pointed towards a compound-specific sensitivity of mice and derived cells lacking p53.S389 phosphorylation. To get better knowledge on this compound sensitivity we performed here additional studies with an extended set of compounds. Questions addressed were the following:
Does a specific class of DNA-damaging agents trigger p53.S389 phosphorylation?
Exposure to UV, 2-AAF and MNU all resulted in the rapid induction of total p53 protein levels as well as p53.S389 phosphorylation. However, clear differences were detectable between p53 induction and levels of p53 phosphorylated at serine 389 after exposure to the different DNA damaging agents. Total p53 protein levels were higher in cells exposed to UV or 2-AAF compared to MNU and, furthermore, exposure to UV resulted in more abundant p53.S389 phosphorylation levels as compared to 2-AAF. Although compounds leading to DNA damage that is substrate to NER seem to induce p53 more efficiently, we have to conclude that p53.S389 phosphorylation is not restricted to agents introducing DNA damage that is substrate to a specific repair pathway (NER). Still though, we cannot exclude that certain compounds have broader lesion spectra, including those to be repaired through NER, than we currently assume.
Does p53.S389 phosphorylation direct a specific p53-dependent cellular response?
In the present study we showed that p53-dependent induction of apoptosis was exclusively seen after exposure to UV, whereas exposure to 2-AAF resulted in a p53-independent apoptotic response. MNU did not result in apoptotic cells at all. The cell cycle arrest responses which are induced after exposure to UV or 2-AAF appeared to be independent of functional p53.
Surprisingly, exposure to paraquat did not result in any p53 induction or cellular response. Still though, in studies performed in human lung epithelial-like cells [58] it was found that paraquat induced p53-dependent G1 arrest and apoptosis. Apparently, MEFs are not very suitable in detecting p53-driven cellular responses upon DNA damage, with the notable exception of UV-induced processes. Possibly, DNA repair is so efficient or MEFs are not sensitive to DNA damage, thereby causing that p53-dependent defense mechanisms go undetected in these cells.
Is there an interaction between DNA repair, stalled RNA polymerases and p53.S389 phosphorylation?
It is generally accepted that, in contrast to the human situation, rodent cells have inefficient GG-NER activity. This striking difference is thought to be mainly caused by the fact that DNA damage recognition factors involved in GG-NER, like XPC and XPE, are not p53 responsive in rodents, like they are in human cells [59-61]. In the MEFs used in this study, i.e., wild- type, p53.S389A and p53-/- cells, we also found no differences in overall GG-NER activity (UV-induced UDS, results not shown). This indicates that the effects of diminished p53.S389 phosphorylation, we previously found in vivo (i.e., increased skin tumor development upon UV exposure), is possibly attributable to decreased TC-NER activity. Therefore, we thought that blockage of transcription, might be the responsible trigger for p53.S389 phosphorylation. The connection between blocked RNA polymerase and the induction of p53 activity in response to DNA damage had been described before [33]. The inhibition of transcription elongation, due to DNA damages on the transcribed strand of active genes, was furthermore linked to post- translational modifications at different p53 sites (like Ser15 and Lys382) [35]. In this study we used MEFs with complementary defects in the NER sub-pathways (Figure 3) and we concluded
that phosphorylation of p53.S389 is additionally linked to hampered TC-NER activity. Indeed Xpa-/- as well as Csb-/- MEFs, both deficient in TC-NER, rapidly accumulated high p53 and phosphorylated p53.S389 levels as compared to wild-type and Xpc-/- cells.
All these results point towards the notion that p53.S389 phosphorylation is linked to hampered elongation of transcription. This was also substantiated by the finding that doxorubicin, also described as an inhibitor of RNA transcription [46;62;63], also led to p53.S389 phosphorylation.
Since doxorubicin is also known as an inhibitor of DNA polymerases, we cannot exclude that blocked DNA replication may also lead to p53.S389 phosphorylation [46;64;65].
However, when it would be solely an effect of inhibited DNA polymerases, then one would expect that p53 levels in wild-type, Xpa-/-,Csb-/-, and Xpc-/- are the same like those found upon doxorubicin exposure (Figure 4). In contrast to that, it appeared that Xpa-/- and Csb-/- MEFs, both lacking TC-NER, showed a differential and more robust induction of p53.S389 phosphorylation after exposure to UV as compared to those found in wild-type and Xpc-/- MEFs.
We, therefore, believe that phosphorylation of p53.S389 is mainly driven by stalled RNA- polymerases. Unfortunately, we still do not know the ultimate cellular response of a cell upon p53.S389 phosphorylation. In MEFs and upon exposure to UV the p53.S389 phosphorylation clearly stimulates the p53-dependent apoptosis. For other compounds and measured in other cellular systems this situation is clearly more complex and needs further research.
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