HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 39, 36425-36430, September 28, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/39/36425    most recent M105215200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mendoza-Alvarez, H. Articles by Alvarez-Gonzalez, R. Search for Related Content PubMed PubMed Citation Articles by Mendoza-Alvarez, H. Articles by Alvarez-Gonzalez, R. Social Bookmarking                      What's this?

Regulation of p53 Sequence-specific DNA-binding by Covalent Poly(ADP-ribosyl)ation^*

Hilda Mendoza-Alvarez and Rafael Alvarez-Gonzalez

From the Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth, Fort Worth, Texas 76107

Received for publication, June 6, 2001, and in revised form, July 17, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have characterized the covalent poly(ADP-ribosyl)ation of p53 using an in vitro reconstituted system. We used recombinant wild type p53, recombinant poly(ADP-ribose) polymerase-1 (PARP-1) (EC 2.4.2.30), and NAD⁺. Our results show that the covalent poly(ADP-ribosyl)ation of p53 is a time-dependent protein-poly(ADP-ribosyl)ation reaction and that the addition of this tumor suppressor protein to a PARP-1 automodification mixture stimulates total protein-poly(ADP-ribosyl)ation 3- to 4-fold. Electrophoretic analysis of the products synthesized indicated that short oligomers predominate early during hetero-poly(ADP-ribosyl)ation, whereas longer ADP-ribose chains are synthesized at later times of incubation. A more drastic effect in the complexity of the ADP-ribose chains generated was observed when the NAD⁺ concentration was varied. As expected, increasing the NAD⁺ concentration from low nanomolar to high micromolar levels resulted in the slower electrophoretic migration of the p53-(ADP-ribose)_n adducts. Increasing the concentration of p53 protein from low nanomolar (40 nM) to low micromolar (1.0 µM) yielded higher amounts of poly(ADP-ribosyl)ated p53 as well. Thus, the reaction was acceptor protein concentration-dependent. The hetero-poly(ADP-ribosyl)ation of p53 also showed that high concentrations of p53 specifically stimulated the automodification reaction of PARP-1. The covalent modification of p53 resulted in the inhibition of the binding ability of this transcription factor to its DNA consensus sequence as judged by electrophoretic mobility shift assays. In fact, controls carried out with calf thymus DNA, NAD⁺, PARP-1, and automodified PARP-1 confirmed our conclusion that the covalent poly(ADP-ribosyl)ation of p53 results in the transcriptional inactivation of this tumor suppressor protein.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The covalent poly(ADP-ribosyl)ation of DNA-binding proteins in eucaryotes is a post-translational modification reaction that has been implicated in the modulation of chromatin structure and function in DNA-damaged and apoptotic cells (1-3). The immediate synthesis of poly(ADP-ribose) from NAD⁺ in response to DNA strand break formation in vivo is mostly catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1)¹ (1-3). This enzyme was believed for some time to be the only nuclear DNA-dependent enzyme (EC 2.4.2.30) responsible for the synthesis of chromatin-bound ADP-ribose chains (1-3). However, over the last 4 years, and since the last International Symposium on protein-poly(ADP-ribosyl)ation (4), other novel and less abundant PARP-like proteins have been identified and reported (5-8). The physiological existence of other proteins with ADP-ribose-polymerizing activity explains why PARP-1 (/) knockout cells still display a positive immunofluorescent nuclear signal when exposed to a fluorescently tagged monoclonal antibody specific for this unique nucleic acid (9). Nevertheless, it appears that about 90% of the total protein-bound polymers synthesized in DNA-damaged cells are assembled by PARP-1. Although most of these genotoxicity-dependent polymers of ADP-ribose seem to be covalently bound to PARP-1 itself (10-13), other chromatin proteins, including histones (14, 15), DNA-metabolizing enzymes (16, 17), and transcription factors (18-20), have been reported to be covalent targets for poly(ADP-ribosyl)ation. In this report, we have focused on the biochemical characterization of p53 poly(ADP-ribosyl)ation as well as the functional consequences of the substantial covalent modification of this tumor suppressor protein on its DNA-binding properties.

Tumor suppressor p53 is an inducible protein that accumulates in the nucleus following DNA damage. Interestingly, the increased expression of p53 has recently been shown to initially parallel the expression of PARP-1 in high grade lymphomas (21), and these events apparently precede the enzymatic activation of the latter in DNA-damaged cells (19). Not surprisingly, both p53 (22) and PARP-1 (23) have been "classified" as guardians of the eucaryotic genome. In fact, both DNA-binding proteins have been shown to physically associate via specific protein-protein interactions in genotoxically treated cells (19, 24, 25). The molecular association between these DNA damage protein sensors may actually result in the NAD⁺-dependent covalent poly(ADP-ribosyl)ation of the tumor suppressor protein both in vitro (19, 24) and in vivo (26). However, the functional consequences of the covalent poly(ADP-ribosyl)ation of p53 as a transcription factor remain to be elucidated.

It should be mentioned that the tumor suppressor p53 functions as a cell cycle checkpoint in maintaining genomic stability in mammals. In fact, over half of most human cancers contain mutations in the p53 tumor suppressor gene and over 90% of the p53 missense mutations are clustered within the sequence-specific DNA-binding domain. Overwhelming experimental evidence suggests that the functional inactivation of p53, especially its DNA-binding activity, is a crucial, and often obligatory step in the complex process of tumorigenesis. By contrast, p53 is either induced or activated in response to a plethora of stimuli, including DNA damage. The activation of p53 leads to one of two major cellular pathways: apoptosis or cell cycle arrest at the G₁ phase preventing progression through the S phase until damaged DNA is repaired (27). Although the transcriptional properties of p53 are well established in cell cycle arrest following DNA damage, this is not necessarily so in p53-mediated apoptosis.

The tumor suppressor protein p53 may also function as a repressor of a variety of viral and cellular gene promoters that lack p53 binding sites (28-30), presumably via its carboxyl-terminal fragment (31). Nevertheless, it is clear that the main function of this tumor suppressor protein is as a transcription factor in which p53 up-regulates specific cell cycle arrest-related genes (32, 33).

As a transcription factor, p53 recognizes a specific consensus DNA sequence consisting of two copies of the 10-bp motif, 5'-PuPuPuC(A/T)(T/A)GpyPyPy-3', separated by a 0- to 13-bp spacer. Wild type p53 transactivates the expression of specific target genes by specifically binding to p53-binding sites in the sequences of these genes (32, 33). However, little is known about what kind of biochemical signals regulate the sequence-specific DNA binding affinity of p53. Because p53 has been shown to physically interact with PARP-1 (19) and ADP-ribose polymers (34) in DNA-damaged cells (see above), we have proceeded to accomplish the following goals. First, to biochemically characterize poly(ADP-ribosyl)ated p53 and second, to determine the ability of poly(ADP-ribosyl)ated p53 to bind to its consensus DNA sequence by electrophoretic mobility shift assays (EMSA). Our results clearly demonstrate the extensive poly(ADP-ribosyl)ation of wild type p53 as a function of: (i) the time of incubation, (ii) the NAD⁺ substrate concentration, and (iii) the p53 protein concentration. Furthermore, we also demonstrate that poly(ADP-ribosyl)ated p53 does not efficiently bind to its consensus DNA sequence by EMSA.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Electrophoresis molecular weight markers and reagents were purchased from Bio-Rad (Hercules, CA); [adenylate-³²P]NAD (specific activity 500 Ci/mmol) was obtained from ICN Biomedicals (Costa Mesa, CA); T4 polynucleotide kinase was purchased from USB Corp. (Cleveland, OH); [-³²P]ATP (specific activity 6000 Ci/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA); wild type recombinant p53 and the consensus binding motif for wt-p53 contained in the oligodeoxynucleotide 5'-TACAGAACATGTCTAAGCATGCTGGGGACT3' and its complimentary strand were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other chemicals were of the highest purity commercially available.

Enzyme Purification-- Poly(ADP-ribose)polymerase-1 (PARP-1) was purified to homogeneity as previously described (35, 50).

5'-End -³²P Radiolabeling of the p53 Consensus Oligodeoxynucleotide Sequence-- A 50-µl incubation reaction mixture containing T4 polynucleotide kinase buffer and unlabeled oligodeoxynucleotide were incubated with 200 pmol of [-³²P]ATP in the presence of T4 polynucleotide kinase for 30 min at 37 °C. The product was purified by a series of precipitations and extractions with 330 mM ammonium acetate and 1% ice-cold ethanol. The final radiospecificity of the product was 6 µC/pmol as determined by scintillation counting.

Poly(ADP-ribosyl)ation of wt-p53-- Triplicate samples containing the required concentrations of wt-p53 were incubated with 18 nM PARP-1 in the presence of 100 mM Tris HCl, pH 8.0, 10 mM MgCl, 1 mM dithiothreitol as well as [³²P]NAD⁺ and activated calf thymus DNA at 37 °C as indicated in the corresponding figure legend. 50 µl of SDS-loading buffer was added to one of each triplicate sample and loaded onto a 4-15% polyacrylamide gel. The poly(ADP-ribosyl)ated proteins were detected by autoradiographic analysis of the dried gel. Two samples from each triplicate were precipitated with 20% (w/v) trichloroacetic acid, washed, and counted to determine the total incorporation of ADP-ribose under each distinct set of conditions. One of the duplicate samples was used to determine the ADP-ribose polymer size distribution by high resolution polyacrylamide gel electrophoresis (see below). A final concentration of 5 µM NAD⁺ was used for the synthesis of unlabeled ADP-ribose polymers as necessary to analyze the effect of p53 poly(ADP-ribosyl)ation on DNA binding.

Size Distribution of ADP-ribose Chains by High Resolution Polyacrylamide Gel Electrophoresis-- Acid-precipitable material was processed for analysis of the ADP-ribose chains as previously published (35). Briefly, the ADP-ribose polymers were chemically detached from protein acceptor with 0.1 N NaOH and 20 mM EDTA for 2 h at 60 °C, neutralized, and diluted in 60 mM tris borate-EDTA (TBE) buffer, pH 8.3. Protein-free ADP-ribose polymers were then subjected to electrophoresis on a (20 × 20 cm) 20% polyacrylamide gel. The size distribution of the ADP-ribose chains was visualized following overnight autoradiographic exposure to Kodak (Biomax-MR) film.

Determination of Wild Type p53 Sequence-specific DNA Binding by Electrophoretic Mobility Shift Assays-- The effect of the poly(ADP-ribosyl)ation of wt-p53 on the binding to its ³²P-radiolabeled consensus oligodeoxynucleotide sequence was determined by electrophoretic mobility shift assays as reported elsewhere (36) with the following modifications. First, either native or poly(ADP-ribosyl)ated p53 were incubated with 0.4 ng of -³²P-radiolabeled oligodeoxynucleotide probe in the presence of 100 mM Tris-HCl, pH 8, 25 mM KCl, 0.025% Nonidet P-40, 0.1 mg/ml bovine serum albumin, 0.1 mM EDTA, and 5% glycerol for 15 min at 37 °C. Immediately following incubation, samples were loaded onto a native 4-20% acrylamide-TBE gradient gel and electrophoresed in TBE buffer at 150 V for ~60 min at 4 °C. Binding of wt-p53 to its consensus DNA sequence (20-mer) was analyzed by autoradiography following exposure of the dried gel to x-ray film.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recently, we (19), as well as others (24-26), have shown the physical association of p53 with PARP-1 in DNA-damaged and apoptotic cells. However, this protein-protein association usually does not result in the covalent poly(ADP-ribosyl)ation of the tumor suppressor protein (19, 24, 25). In fact, the covalent poly(ADP-ribosyl)ation of p53 only takes place under specific conditions, e.g. when PARP-1 has not yet been proteolyzed by caspases 3 or 7 in apoptotic cells. That is why we could only confirm the covalent poly(ADP-ribosyl)ation of p53 in an apoptotic HeLa cell extract (19) after calf thymus PARP-1 was exogenously added. To gain further insight into the physiological significance of p53 poly(ADP-ribosyl)ation in DNA-damaged and apoptotic cells, we decided to biochemically characterize the enzymology of this reaction and its consequences in the sequence-specific DNA binding properties of this tumor suppressor protein.

The Covalent Poly(ADP-ribosyl)ation of Human wt-p53 is Time-dependent-- To confirm that wild type p53 is a covalent target for protein-poly(ADP-ribosyl)ation, the tumor suppressor protein was incubated with 18 nM PARP-1, and 200 nM [³²P]NAD⁺ under the conditions described under "Experimental Procedures." Fig. 1A shows the amount of ADP-ribose incorporated as a function of the time of incubation from 0 to 120 min of incubation. We reproducibly observed that the level of protein-poly(ADP-ribosyl)ation was 3- to 4-fold higher in the presence of wt-p53 (circles) than in its absence (squares), suggesting that not only was PARP-1 efficiently auto-poly(ADP-ribosyl)ating but that the tumor suppressor protein was also covalently modified. To confirm the covalent association of ADP-ribose polymers with wt-p53, we carried out SDS-polyacrylamide gel electrophoresis through a 4-15% acrylamide gradient gel. Fig. 1B shows the Coomassie Blue-stained gel where the relative migration of wt-p53 (162 nM) is clearly indicated to the left of the gel. Under these staining conditions, the 100 ng of PARP-1 (18 nM) utilized were not sufficient for strong staining. By contrast, Fig. 1C illustrates the signals developed upon autoradiographic exposure of the dried gel. Lanes 1-7 show the increase in p53 radiolabeling as the time of incubation was extended from 0 to 120 min. Fig. 1C also clearly shows that, although wt-p53 was clearly covalently radiolabeled as a function of time, the efficiency of PARP-1 automodification also increased to the point where the intensity of the PARP-1 band significantly expanded as a result of hyper-poly(ADP-ribosyl)ation. Therefore, it appeared as if the modification of wt-p53 increased the efficiency of PARP-1 automodification (see below).

View larger version (22K): [in this window] [in a new window]   Fig. 1.   The covalent poly(ADP-ribosyl)ation of wt-p53 is time-dependent. A, graphical increase of ADP-ribose covalently incorporated (picomoles) onto protein in the absence (squares) or presence (circles) of 81 nM p53 and 200 nM NAD+ as indicated under "Experimental Procedures." B, Coomassie Blue staining of wt-p53 after incubation with 18 nM PARP-1 and 200 nM NAD+ for 0, 2.5, 5, 15, 30, 60, and 120 min. C, corresponding autoradiograph of the gel shown on B. In both cases the relative electrophoretic migration of PARP-1 and p53 is shown to the left. D, distribution of 32P-radiolabeled polymers by high resolution polyacrylamide gel electrophoresis following chemical release from protein as indicated under "Experimental Procedures." The relative electrophoretic migrations of AMP (monomers), BPB [(ADP-ribose)8], and XC [(ADP-ribose)20] are shown to the left of D. The times of incubation, reactants present, and other specific incubation conditions are listed at the bottom of D for each specific set of conditions (lanes 1-11).

Due to the dramatic change in the electrophoretic behavior of auto-poly(ADP-ribosyl)ated-PARP-1, we next decided to determine the size distribution of the protein-bound ADP-ribose polymers. We accomplished this by high resolution polyacrylamide gel electrophoresis. Samples were processed after chemical release from protein under alkaline conditions in the presence of EDTA (35). Fig. 1D shows the size distribution of the protein-free ADP-ribose chains synthesized when PARP-1 (18 nM) and wt-p53 (81 nM) were co-incubated as a function of time (see above). Lanes 1, 2, and 3 show the mono(ADP-ribosyl)ation step of ADP-ribose polymer synthesis predominated in the first 5 min of incubation (AMP band). Also shown are ADP-ribose chains of up to 20 ADP-ribose units (lane 4) or more (lanes 5-7), which represent the polymers synthesized at 1 and 2 h of incubation. Lanes 8, 10, and 11 show the absence of protein-bound ADP-ribose polymers when DNA, NAD⁺, or PARP-1 was omitted from the incubation mixture. By contrast, lane 9 shows the polymer size distribution of the enzyme products generated in the absence of wt-p53. Although the distribution of polymers was very similar without (lane 9) and with (lanes 5-7) wt-p53, the overall yield of (ADP-ribose)_n-protein adducts was consistently higher in the presence of the tumor suppressor protein.

The Covalent Poly(ADP-ribosyl)ation of Human wt-p53 is NAD⁺ Concentration-dependent-- Recently, we have reported (37) that the size distribution of the enzyme-bound ADP-ribose polymers synthesized during the automodification reaction is determined by the concentration of NAD⁺ in the assay regardless of the protein concentration in the incubation mixture. Therefore, we next proceeded to determine the effect that increasing the NAD⁺ concentration would have on the electrophoretic mobility of poly(ADP-ribosyl)ated-PARP-1 and poly(ADP-ribosyl)ated p53. Fig. 2A shows the significant decrease in the Coomassie Blue staining intensity of the wt-p53 (250 nM) band as the concentration of NAD⁺ was increased from 0-1 µM (lanes 1-4) to 100-1000 µM (lanes 5-7). The remarkable decrease in staining intensity of this protein is apparently due to the increased levels of wt-p53 poly(ADP-ribosyl)ation at more physiological levels of NAD⁺, namely 0.5-1.0 mM substrate concentration, because under these conditions, the protein adducts stay at the top of the gel. Our interpretation was later confirmed by autoradiographic analysis of the same gel upon exposure to x-ray film. As expected, Fig. 2B shows that the electrophoretic migration of auto-poly(ADP-ribosyl)ated-(PARP-1) significantly decreased from the typical 113-kDa position (lane 2) at 250 nM NAD⁺, to the origin of the gel (lanes 4-7) at micromolar levels of NAD⁺ (10, 100, 500, and 1000 µM, respectively). Therefore, we conclude that the molecules of wt-p53 that become covalently modified transform into heavily poly(ADP-ribosyl)ated protein adducts that do not migrate into the gel, just like hyper-poly(ADP-ribosyl)ated-PARP-1.

View larger version (60K): [in this window] [in a new window]   Fig. 2.   The covalent poly(ADP-ribosyl)ation of wt-p53 is NAD+-concentration dependent. A, Coomassie Blue staining of wt-p53 after incubation with 18 nM PARP-1 for 15 min at 37 °C and 0, 0.25, 0.5, 1.0, 10, 100, 500, and 1000 µM NAD+ (lanes 1-8, respectively). B, corresponding autoradiograph of the gel shown in A. In both cases the relative electrophoretic migration of PARP-1 and wt-p53 is shown to the left. The electrophoretic migration of the molecular weight markers is shown to the right of A and B.

To our knowledge, human wt-p53 (this report) and TFIIF (18), are the only transcription factors that have been carefully characterized in terms of the biochemistry of protein-poly(ADP-ribosyl)ation. Thus, to evaluate the influence of all molecular components in the protein-poly(ADP-ribosyl)ation mixture, we next determined the effect of p53 protein concentration in the modification of this tumor suppressor protein.

The Covalent Poly(ADP-ribosyl)ation of Human wt-p53 Is Tumor Suppressor Protein Concentration-dependent-- Fig. 3 illustrates the effect of p53 protein concentration on its poly(ADP-ribosyl)ation catalyzed by PARP-1 (18 nM) and NAD⁺ (200 nM) after 30 min of incubation at 37 °C. Fig. 3A shows the Coomassie Blue-stained gel of this experiment as the wt-p53 protein concentration was increased from 0 to 1000 nM (lanes 1-7). Lanes 4 through 7 represent 160 nM, 250 nM, 500 nM, and 1.0 µM, respectively. These levels of p53 concentration were the only ones that contained enough protein for Coomassie Blue staining. Fig. 3B shows the autoradiograph of the same gel. We observed that, although a ratio of 2:1 of p53/PARP-1 was not enough for the ADP-ribose polymer modification of these polypeptides (Fig. 3B, lane 2), a ratio of at least 4:1 p53/PARP-1 was required for a positive signal (Fig. 3B, lane 3). Needless to say that higher concentrations of p53 (lanes 4-7) resulted in a stronger protein-poly(ADP-ribosyl)ation signal. Interestingly, although the NAD⁺ concentration remained constant (200 nM), the efficiency of the PARP-1 automodification reaction increased as the total amount of wt-p53 in the incubation reaction mixture was elevated (compare the thickness of the PARP-1 radiolabeled band on lanes 1 and 7).

View larger version (38K): [in this window] [in a new window]   Fig. 3.   The covalent poly(ADP-ribosyl)ation of wt-p53 is tumor suppressor protein-concentration dependent. A, Coomassie Blue staining of wt-p53 after incubation with 18 nM PARP-1 and 200 nM NAD+ for 30 min at 37 °C. Lanes 1 through 8 correspond to individual protein-poly(ADP-ribosyl)ation incubations with 0, 40, 80, 160, 250, 500, and 1000 nM wt-p53, respectively. B, corresponding autoradiograph of the gel shown in A. In both cases the relative electrophoretic migration of PARP-1 and wt-p53 is shown to the left. The electrophoretic migration of the molecular weight markers is shown to the right of A and B.

Once we reproducibly confirmed the covalent poly(ADP-ribosyl)ation of human wt-p53, we proceeded to determine its effect on the sequence-specific DNA binding of this tumor suppressor protein by EMSA.

Comparison of Sequence-specific DNA Binding between Native wt-p53 and Covalently Poly(ADP-ribosyl)ated p53 by EMSA-- Fig. 4 shows the EMSA analysis of both native p53 and poly(ADP-ribosyl)ated p53 upon incubation with its ³²P-radiolabeled oligodeoxynucleotide consensus DNA sequence. Lane 1 displays the electrophoretic migration of the oligodeoxynucleotide probe alone. Lane 2 shows the mobility shift caused by the addition of 400 ng of wt-p53. Lanes 3, 4, and 5 display the effect of individual protein-poly(ADP-ribosyl)ation reaction components on the wt-p53 DNA sequence-specific mobility shift. Although addition of NAD⁺, the ADP-ribosylation substrate alone, did not affect the mobility shift signal (lane 3), addition of active calf thymus DNA significantly inhibited the mobility shift (lane 4). This was a fully anticipated result, because p53 is also known to possess nonspecific single-stranded DNA binding properties in its carboxyl-terminal domain (38-40). Therefore, we conclude that the result shown in Fig. 4, lane 4, is simply a competition effect for DNA binding. By contrast, the light inhibition observed upon addition of PARP-1 alone (lane 5) probably reflects the fact that PARP-1itself is a DNA-binding protein that binds to DNA free ends, such as those present at 5' and/or 3' of the oligodeoxynucleotide probe. Presumably, the effect of PARP-1 inhibition is not quantitative, because it does not bind the probe efficiently, even at high nanogram levels (see Fig. 5 below). Surprisingly, addition of all protein-poly(ADP-ribosyl)ation ingredients (Fig. 4, lane 6) caused the strongest inhibition of p53 sequence-specific DNA binding. Therefore, we conclude that the direct covalent poly(ADP-ribosyl)ation of wt-p53, PARP-1, or both directly result in the specific inhibition of wt-p53 binding to its consensus DNA. To distinguish between these possibilities, we also carried out an incubation where active calf thymus DNA was omitted from the incubation mixture (Fig. 4, lane 7). Because omission of activating DNA from the EMSA incubation limits the extent of the automodification reaction of PARP-1, while still allowing the covalent poly(ADP-ribosyl)ation of p53 (41), we could measure the effect of p53 poly(ADP-ribosyl)ation on its sequence-specific DNA binding ability. Fig. 4, lane 7 shows that, under these conditions, there is a significant inhibition of p53 DNA binding (compare with lanes 2 and 6). Omission of NAD⁺ alone (lane 8) significantly reduced the mobility shift of the p53 oligodeoxynucleotide probe as well. However, this effect was 1) mainly due to the presence of active calf thymus DNA and 2) to PARP-1 (compare with lanes 4 and 5). Finally, Fig. 5 shows a weak interaction between PARP-1 and the radiolabeled DNA probe in the absence of calf thymus DNA (see above). In fact, addition of increasing amounts of PARP-1 from 7 to 70 ng did not result in an increased electrophoretic retardation of the DNA probe. Therefore, the addition of PARP-1 would not be expected to quantitatively inhibit the mobility shift caused by wtp53 (see Fig. 4, lane 5 above).

View larger version (64K): [in this window] [in a new window]   Fig. 4.   The sequence-specific DNA binding of human wt-p53 is dependent on the covalent poly(ADP-ribosyl)ation of this tumor suppressor protein. EMSA was performed as described under "Experimental Procedures." First, either native (lane 2) or poly(ADP-ribosyl)ated p53 (lane 6) were incubated with 0.4 ng of -32P-radiolabeled oligodeoxynucleotide probe in the presence of 100 mM Tris-HCl, pH 8, 25 mM KCl, 0.025% Nonidet P-40, 0.1 mg/ml bovine serum albumin, 0.1 mM EDTA, and 5% glycerol for 15 min at 37 °C. Immediately following incubation, samples were loaded onto a native 4-20% acrylamide-TBE gradient gel and electrophoresed in TBE buffer at 150 V for ~60 min at 4 °C. Lane 1, DNA probe control; lanes 3 and 4, effect of either NAD+ or DNA on the p53 mobility shift, respectively; lane 5, effect of PARP-1 alone; lane 6, effect of the addition of PARP-1; and lanes 7 and 8, effect of DNA and NAD+ omission in the DNA binding incubation mixtures, respectively.

View larger version (45K): [in this window] [in a new window]   Fig. 5.   Binding of poly(ADP-ribose) polymerase-1 to the p53 DNA probe does not increase from 7 to 70 ng of PARP-1/incubation. EMSA was performed as described under "Experimental Procedures." Lane 1, electrophoretic migration of 0.4 ng of the -32P-radiolabeled oligodeoxynucleotide p53 consensus sequence alone. Lanes 2-6, electrophoretic effect of 7, 14, 28, 56, and 70 ng of PARP-1 on the p53 DNA probe. Samples were loaded onto a native 4-20% acrylamide-TBE gradient gel and electrophoresed in TBE buffer at 150 V for ~60 min at 4 °C.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The tumor suppressor protein p53 is thought to function as a cell cycle checkpoint to maintain genomic stability in mammals. As a result it may also be considered the "ultimate gatekeeper." A master molecule deciding the ultimate fate of the cell after substantial levels of DNA damage, e.g. "survival" or "cell death." Not surprisingly, more than 50% of human tumors, especially those of lung, colon, liver, and breast cancer, may contain mutations in the p53 gene. In fact, over 90% of the p53 missense mutations are typically clustered within its sequence-specific DNA-binding domain. Thus, overwhelming experimental evidence suggests that the functional activation of p53, especially the DNA-binding activity of p53, is a crucial, and often obligatory step to prevent the development of tumors. The molecular activation of p53 may usually lead to one of two major physiological pathways: programmed cell death or cell cycle arrest. Therefore, any molecular signal that turns "on" or "off" the DNA-binding properties of p53 either as a transcription factor (32, 33) or as a genetic repressor (27-31) would be of significant interest to study. Frequently, the physiological function of crucial metabolic proteins is regulated by covalent post-translational modification. Not surprisingly, Hupp et al. (42) first proposed that the DNA-binding activity of p53 might be regulated by the specific phosphorylation of the carboxyl-terminal DNA-binding domain. As we discussed above, it was later found (39-40) that the carboxyl-terminal peptide of p53 was specific for binding single-stranded DNA, which in turn may be related to the potential function(s) of p53 as a gene repressor (27-31) rather than as a transcription factor (32, 33). Nevertheless, others (43) have reported that the phosphorylation of p53 at other peptide sites may lead to p53-dependent transcriptional attenuation. However, it was not clear whether the observed reduction in p53-dependent transcriptional responses was due to the inability of this protein to oligomerize as a tetramer (43). Indeed, tetrameric p53 has previously been shown to be the latent and active form of this tumor suppressor gene (44). Alternatively, the phosphorylation of the p53 carboxyl terminus might actually reduce the sequence-specific DNA binding of this transcription factor via conformational changes that may affect the ionic interactions of its central domain with DNA itself. By contrast, the phosphorylation of three serine residues at the amino terminus of p53 (serines 9, 18, and 37) was also reported to facilitate p53 transcriptional function (45). However, the kinase modification of these sites did not change the intracellular localization, oligomerization, and DNA-binding properties of p53 (45). Therefore, it appears that this type of post-translational modification of p53 is not the direct post-translational mechanism to reversibly regulate its transactivating properties. Recently, it was also demonstrated that the sequence-specific DNA binding of this tumor suppressor protein was activated by the acetylation of its carboxyl-terminal domain (46). Thus, it seems that both phosphorylation and acetylation target sites on p53 localized to either the first 100 amino acids of its amino terminus or the last 90 amino acids of its carboxyl terminus (47-49).

Here, we concentrated on the biochemical characterization of the covalent poly(ADP-ribosyl)ation of p53. The massive addition of ADP-ribose polymers of over 20 units (Fig. 1D) immediately suggests that the addition of over 40 negative charges (two for every ADP-ribose unit) should result in the ionic repulsion between post-translationally modified p53 and its consensus sequence. In fact, the significant reduction in the electrophoretic migration of poly(ADP-ribosyl)ated p53 adducts (Fig. 2) generated at physiological concentrations of NAD⁺ (e.g. 500-1000 µM) strongly suggested to us that the protein-protein interactions of p53 with PARP-1 (19, 24-26) may result in the reversible inhibition of its sequence-specific DNA binding. Not surprisingly, we reproducibly observed that p53 lost its DNA-binding properties upon covalent poly(ADP-ribosyl)ation (Fig. 4). We are currently in the process of identifying the poly(ADP-ribosyl)ation amino acid sites on both wild type and mutant p53 molecules to determine whether they are actually localized on its centrally located sequence-specific DNA-binding domain, its single-stranded DNA carboxyl-terminal binding domain, or both. Our results are also in agreement with the data of Malanga and Althaus (34) who demonstrated that the presence of protein-free and highly branched polymers of ADP-ribose in a p53 mobility shift assay resulted in the strong inhibition of sequence-specific DNA binding. Although ADP-ribose polymers are never protein-free in situ, proteins susceptible to covalent modification with highly branched polymers may be responsible for the reversible regulation of p53-dependent transactivation. Even though it has been clearly demonstrated that highly branched polymers are covalently bound to PARP-1 itself (10-12, 15-19, 35, 37) and not to histones (14-16), we report here for the first time, that highly complex ADP-ribose polymers can also be covalently bound to p53 (Fig. 2). Furthermore, we also show that the strong protein-protein association of p53 with PARP-1 in vitro (19) and in vivo (24-26) my lead to the higher efficiency of PARP-1 automodification shown in Figs. 1 and 3. Recently, we showed that the automodification reaction of PARP-1 directly activates the sequence-specific DNA binding of NF-B (50), a well-established anti-apoptotic transcription factor. Therefore, low levels of DNA damage in cultured cells would presumably lead to the initial non-covalent recruitment of p53 (34) to DNA-damaged sites to allow for cell cycle arrest, DNA repair, and cell survival. By contrast, under high levels of DNA damage, the covalent poly(ADP-ribosyl)ation of the tumor suppressor protein would inhibit cell cycle arrest and p53-dependent gene expression (27, 28). With the inhibition of the p53 sequence-specific DNA binding and the caspase-catalyzed degradation of PARP-1, severely DNA-damaged cells would initiate the execution phase of the cell death program (13). Finally, one should also keep in mind that, under specific conditions, NF-B may actually facilitate programmed cell death and could be essential for p53-mediated apoptosis (51). Therefore, a better understanding of the interplay between NF-B and p53 with regards to the activity of PARP-1 in programmed cell death needs to be evaluated in vivo as well.

	ACKNOWLEDGEMENTS

We thank Dr. Hanswalter Zentgraf, Institute for Tumor Virology, German Cancer Research Center, Heidelberg, Germany for his gift of the baculovirus and E. coli recombinant p53 proteins used in some of the control experiments in this study.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM45451 (to R. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107. Tel.: 817-735-2117; Fax: 817-735-2133; E-mail: ralvarez@hsc.unt.edu.

Published, JBC Papers in Press, July 26, 2001, DOI 10.1074/jbc.M105215200

	ABBREVIATIONS

The abbreviations used are: PARP-1, poly(ADP-ribose) polymerase-1; bp, base pair(s); EMSA, electrophoretic mobility shift assay.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				S. Beneke, O. Cohausz, M. Malanga, P. Boukamp, F. Althaus, and A. Burkle Rapid regulation of telomere length is mediated by poly(ADP-ribose) polymerase-1 Nucleic Acids Res., October 3, 2008; (2008) gkn615v1. [Abstract] [Full Text] [PDF]

				F. Kappes, J. Fahrer, M. S. Khodadoust, A. Tabbert, C. Strasser, N. Mor-Vaknin, M. Moreno-Villanueva, A. Burkle, D. M. Markovitz, and E. Ferrando-May DEK Is a Poly(ADP-Ribose) Acceptor in Apoptosis and Mediates Resistance to Genotoxic Stress Mol. Cell. Biol., May 15, 2008; 28(10): 3245 - 3257. [Abstract] [Full Text] [PDF]

				O. A. Olabisi, N. Soto-Nieves, E. Nieves, T. T. C. Yang, X. Yang, R. Y. L. Yu, H. Y. Suk, F. Macian, and C.-W. Chow Regulation of Transcription Factor NFAT by ADP-Ribosylation Mol. Cell. Biol., May 1, 2008; 28(9): 2860 - 2871. [Abstract] [Full Text] [PDF]

				J. Fahrer, R. Kranaster, M. Altmeyer, A. Marx, and A. Burkle Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length Nucleic Acids Res., December 18, 2007; 35(21): e143 - e143. [Abstract] [Full Text] [PDF]

				R. Alvarez-Gonzalez Genomic Maintenance: The p53 Poly(ADP-ribosyl)ation Connection Sci. Signal., December 4, 2007; 2007(415): pe68 - pe68. [Abstract] [Full Text] [PDF]

				S. Beneke and A. Burkle Poly(ADP-ribosyl)ation in mammalian ageing Nucleic Acids Res., December 3, 2007; 35(22): 7456 - 7465. [Abstract] [Full Text] [PDF]

				C. Keil, T. Grobe, and S. Li Oei MNNG-induced Cell Death Is Controlled by Interactions between PARP-1, Poly(ADP-ribose) Glycohydrolase, and XRCC1 J. Biol. Chem., November 10, 2006; 281(45): 34394 - 34405. [Abstract] [Full Text] [PDF]

				C. Baumann, G. S. Boehden, A. Burkle, and L. Wiesmuller Poly(ADP-RIBOSE) polymerase-1 (Parp-1) antagonizes topoisomerase I-dependent recombination stimulation by P53 Nucleic Acids Res., February 9, 2006; 34(3): 1036 - 1049. [Abstract] [Full Text] [PDF]

				J.-Y. Huang, W.-H. Chen, Y.-L. Chang, H.-T. Wang, W.-t. Chuang, and S.-C. Lee Modulation of nucleosome-binding activity of FACT by poly(ADP-ribosyl)ation. Nucleic Acids Res., January 1, 2006; 34(8): 2398 - 2407. [Abstract] [Full Text] [PDF]

				M. D. Vodenicharov, M. M. Ghodgaonkar, S. S. Halappanavar, R. G. Shah, and G. M. Shah Mechanism of early biphasic activation of poly(ADP-ribose) polymerase-1 in response to ultraviolet B radiation J. Cell Sci., February 1, 2005; 118(3): 589 - 599. [Abstract] [Full Text] [PDF]

				L. Zheng, C. Szabo, and T. S. Kern Poly(ADP-Ribose) Polymerase Is Involved in the Development of Diabetic Retinopathy via Regulation of Nuclear Factor-{kappa}B Diabetes, November 1, 2004; 53(11): 2960 - 2967. [Abstract] [Full Text] [PDF]

				H. Nakajima, H. Nagaso, N. Kakui, M. Ishikawa, T. Hiranuma, and S. Hoshiko Critical Role of the Automodification of Poly(ADP-ribose) Polymerase-1 in Nuclear Factor-{kappa}B-dependent Gene Expression in Primary Cultured Mouse Glial Cells J. Biol. Chem., October 8, 2004; 279(41): 42774 - 42786. [Abstract] [Full Text] [PDF]

				S. Wieler, J.-P. Gagne, H. Vaziri, G. G. Poirier, and S. Benchimol Poly(ADP-ribose) Polymerase-1 Is a Positive Regulator of the p53-mediated G1 Arrest Response following Ionizing Radiation J. Biol. Chem., May 23, 2003; 278(21): 18914 - 18921. [Abstract] [Full Text] [PDF]

				L. Virag and C. Szabo The Therapeutic Potential of Poly(ADP-Ribose) Polymerase Inhibitors Pharmacol. Rev., September 1, 2002; 54(3): 375 - 429. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 276/39/36425    most recent M105215200v1 Alert me when this article is cited