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Phosphorylation of p53 on Key Serines Is Dispensable for Transcriptional Activation and Apoptosis

2004; Elsevier BV; Volume: 279; Issue: 51 Linguagem: Inglês

10.1074/jbc.m410233200

1083-351X

Thelma Thompson, Christian Tovar, Hong Yang, Daisy Carvajal, Binh Thanh Vu, Qunli Xu, Geoffrey M. Wahl, David Heimbrook, Lyubomir T. Vassilev,

Cancer, Hypoxia, and Metabolism

The p53 tumor suppressor is a key mediator of the cellular response to stress. Phosphorylation induced by multiple stress-activated kinases has been proposed to be essential for p53 stabilization, interaction with transcriptional co-activators, and activation of p53 target genes. However, genetic studies suggest that stress-activated phosphorylation may not be essential for p53 activation. We therefore investigated the role of p53 phosphorylation on six key serine residues (Ser6, Ser15, Ser20, Ser37, Ser46, and Ser392) for p53 activation using nutlin-3, a recently developed small molecule MDM2 antagonist. We show here that nutlin does not induce the phosphorylation of p53. Comparison of the activity of unphosphorylated and phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide in HCT116 and RKO cells revealed no difference in their sequence-specific DNA binding and ability to transactivate p53 target genes and to induce p53-dependent apoptosis. We conclude that p53 phosphorylation on six major serine sites is not required for activation of p53 target genes or biological responses in vivo. The p53 tumor suppressor is a key mediator of the cellular response to stress. Phosphorylation induced by multiple stress-activated kinases has been proposed to be essential for p53 stabilization, interaction with transcriptional co-activators, and activation of p53 target genes. However, genetic studies suggest that stress-activated phosphorylation may not be essential for p53 activation. We therefore investigated the role of p53 phosphorylation on six key serine residues (Ser6, Ser15, Ser20, Ser37, Ser46, and Ser392) for p53 activation using nutlin-3, a recently developed small molecule MDM2 antagonist. We show here that nutlin does not induce the phosphorylation of p53. Comparison of the activity of unphosphorylated and phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide in HCT116 and RKO cells revealed no difference in their sequence-specific DNA binding and ability to transactivate p53 target genes and to induce p53-dependent apoptosis. We conclude that p53 phosphorylation on six major serine sites is not required for activation of p53 target genes or biological responses in vivo. The tumor suppressor p53 is a transcription factor that coordinates a complex network of cellular proteins evolved to protect cells from malignant transformation (1Levine A.J. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6697) Google Scholar, 2Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5744) Google Scholar). In response to diverse stress factors, p53 induces the expression of different subsets of genes leading to cell cycle arrest, apoptosis, DNA repair, or senescence. In cells that are not under stress, p53 activation could suppress their growth or induce apoptosis were it not for the tight regulation elicited by MDM2 (3Freedman D.A. Wu L. Levine A.J. Cell. Mol. Life Sci. 1999; 55: 96-107Crossref PubMed Scopus (480) Google Scholar, 4Michael D. Oren M. Semin. Cancer Biol. 2003; 13: 49-58Crossref PubMed Scopus (640) Google Scholar). The MDM2 gene expression is regulated in part by a p53-responsive promoter. In turn, MDM2 protein binds the p53 N-terminal transactivation domain and negatively regulates tumor suppressor function by compromising transcriptional regulation. Additionally, as MDM2 is an E3 ubiquitin ligase for p53 and itself, MDM2 controls p53 half-life via ubiquitin-dependent degradation. This negative feedback control mechanism assures that both p53 and MDM2 proteins are kept at very low levels in proliferating cells (4Michael D. Oren M. Semin. Cancer Biol. 2003; 13: 49-58Crossref PubMed Scopus (640) Google Scholar).In response to stress, the p53-MDM2 interaction must be disrupted to enable p53 to associate with factors needed for activation of its target genes. Stress-induced p53 activation involves post-translational modification of p53 on multiple sites by phosphorylation, acetylation, and sumoylation (2Vogelstein B. Lane D. Levine A.J. Nature. 2000; 408: 307-310Crossref PubMed Scopus (5744) Google Scholar, 5Brooks C.L. Gu W. Curr. Opin. Cell Biol. 2003; 15: 164-171Crossref PubMed Scopus (630) Google Scholar) and modifications to MDM2 that can enhance MDM2 autoubiquitination and degradation (6Stommel J.M. Wahl G.M. EMBO J. 2004; 23: 1547-1556Crossref PubMed Scopus (312) Google Scholar). With regard to p53 modifications, phosphorylation has been studied most intensively and has been proposed to play a critical role in the stabilization and activation of the tumor suppressor. These studies have been greatly facilitated by the availability of antibodies that recognize p53 modified on specific phosphoserine or phosphothreonine residues (for review, see Ref. 7Apella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (902) Google Scholar). Multiple serine (6, 9, 15, 20, 33, 37, 46, 315, 371, 376, 378, and 392) and three threonine residues (18, 55, and 81) have been reported to undergo phosphorylation in response to diverse stresses. Multiple serine/threonine kinases have been implicated in the upstream signaling leading to p53 phosphorylation (ATM, ATR, DNA-PK, Chk1, Chk2, CK1, p38, CDK2, PKC, JNK), but the precise mechanism of this signaling and its regulation are not well understood (8Jimenez G.S. Khan S.H. Stommel J.M. Wahl G.M. Oncogene. 1999; 18: 7656-7665Crossref PubMed Scopus (167) Google Scholar, 9Ljungman M. Neoplasia. 2000; 2: 208-225Crossref PubMed Scopus (186) Google Scholar). Residues from the N-terminal MDM2 binding domain of p53 (Ser20 and Thr18) have been shown to play a critical role in the interaction between the two proteins and their stress-induced phosphorylation decreases substantially the affinity between p53 and MDM2 when analyzed in vitro using peptide substrates (10Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1013) Google Scholar, 11Craig A.L. Burch L. Vojtesek B. Mikutowska J. Thompson A. Hupp T.R. Biochem. J. 1999; 342: 133-141Crossref PubMed Scopus (124) Google Scholar, 12Schon O. Friedler A. Bycroft M. Freund S.M. Fersht A.R. J. Mol. Biol. 2002; 323: 491-501Crossref PubMed Scopus (269) Google Scholar). These and other studies (7Apella E. Anderson C.W. Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (902) Google Scholar, 13Meek D.W. Oncogene. 1999; 18: 7666-7675Crossref PubMed Scopus (208) Google Scholar) have led to the conclusion that phosphorylation of p53 is a key mechanism responsible for activation of the tumor suppressor in response to cellular stress.In addition to its proposed role in abrogation of p53-MDM2 binding and stabilization of the protein, p53 phosphorylation has also been implicated in regulation of its activity. However, this aspect of p53 phosphorylation is still poorly understood and controversial. Transcriptional activity of p53 is of principal importance for its function as a tumor suppressor. It has been suggested that phosphorylation at specific residues can affect the transcriptional activity of p53 and/or its selectivity toward different subset of genes thus determining the specific type of cellular response to stress (5Brooks C.L. Gu W. Curr. Opin. Cell Biol. 2003; 15: 164-171Crossref PubMed Scopus (630) Google Scholar, 8Jimenez G.S. Khan S.H. Stommel J.M. Wahl G.M. Oncogene. 1999; 18: 7656-7665Crossref PubMed Scopus (167) Google Scholar). Activity of p53 as a transcription factor may be influenced by several factors: (a) ability to form active tetramers, (b) sequence-specific DNA binding, and (c) interaction with other components of the transcriptional machinery. It has been reported that Ser315 and Ser392 phosphorylation may regulate the oligomerization of p53 and thus its sequence-specific DNA binding (14Wang Y. Prives C. Nature. 1995; 376: 88-91Crossref PubMed Scopus (325) Google Scholar, 15Hao M. Lowy A.M. Kapoor M. Deffie A. Liu G. Lozano G. J. Biol. Chem. 1996; 271: 29380-29385Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 16Sakaguchi K. Sakamoto H. Lewis M.S. Anderson C.W. Erickson J.W. Appella E. Xie D. Biochemistry. 1997; 36: 10117-10124Crossref PubMed Scopus (226) Google Scholar). Ser15 phosphorylation has been shown to enhance the interaction of p53 with transcriptional co-activators CBP and PCAF (10Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1013) Google Scholar, 17Lambert P.F. Kashanchi F. Radonovich M.F. Shiekhattar R. Brady J.N. J. Biol. Chem. 1998; 273: 33048-33053Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 18Dumaz N. Meek D.W. EMBO J. 1999; 18: 7002-7010Crossref PubMed Scopus (390) Google Scholar, 19Liu L. Scolnick D.M. Trievel R.C. Zhang H.B. Marmorstein R. Halazonetis T.D. Berger S.L. Mol. Cell. Biol. 1999; 19: 1202-1209Crossref PubMed Scopus (648) Google Scholar). Stress-induced phosphorylation of Ser46 has been implicated in the activation of p53-dependent apoptotic response (20D'Orazi G. Cecchinelli B. Bruno T. Manni I. Higashimoto Y. Saito S. Gostissa M. Coen S. Marchetti A. Del Sal G. Piaggio G. Fanciulli M. Appella E. Soddu S. Nat. Cell Biol. 2002; 4: 11-19Crossref PubMed Scopus (566) Google Scholar, 21Saito S. Goodarzi A.A. Higashimoto Y. Noda Y. Lees-Miller S.P. Appella E. Anderson C.W. J. Biol. Chem. 2002; 277: 12491-12494Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Recently, prolyl isomerase Pin1 has been reported to bind p53 and enhance its DNA binding and transcriptional activity. This binding is dependent on DNA damage-induced phosphorylation of p53 (22Zheng H. You H. Zhou X.Z. Murray S.A. Uchida T. Wulf G. Gu L. Tang X. Lu K.P. Xiao Z.X. Nature. 2002; 419: 849-853Crossref PubMed Scopus (320) Google Scholar, 23Zacchi P. Gostissa M. Uchida T. Salvagno C. Avolio F. Volinia S. Ronai Z. Blandino G. Schneider C. Del Sal G. Nature. 2002; 419: 853-857Crossref PubMed Scopus (357) Google Scholar). Taken together, these observations suggest that p53 phosphorylation may play an important role not only in stabilization of p53 but also in modulation of its transcriptional activity. On the other hand, experiments in which almost all phosphorylation sites in p53 have been mutated demonstrated that transiently expressed phosphorylated and unphosphorylated p53 do not differ significantly in their stability or ability to transactivate reporter genes in p53-null cells (24Ashcroft M. Kubbutat M.H. Vousden K.H. Mol. Cell. Biol. 1999; 19: 1751-1758Crossref PubMed Scopus (376) Google Scholar). Furthermore, studies using mouse mutants with substitutions of Ser15 or Ser20 suggest that these residues are not essential for p53 activation (25Wu Z. Earle J. Saito S. Anderson C.W. Appella E. Xu Y. Mol. Cell. Biol. 2002; 22: 2441-2449Crossref PubMed Scopus (88) Google Scholar, 26Chao C. Hergenhahn M. Kaeser M.D. Wu Z. Saito S. Iggo R. Hollstein M. Appella E. Xu Y. J. Biol. Chem. 2003; 278: 41028-41033Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 27Sluss H.K. Armata H. Gallant J. Jones S.N. Mol. Cell. Biol. 2004; 24: 976-984Crossref PubMed Scopus (121) Google Scholar).Recently, we reported the identification of the first potent and selective small molecule inhibitors of p53-MDM2 interaction, the nutlins (28Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E. Science. 2004; 303: 844-848Crossref PubMed Scopus (3734) Google Scholar). These compounds bind MDM2 in the p53 binding pocket with high selectivity and can release p53 from negative control leading to effective stabilization of p53 and activation of the p53 pathway in vitro and in vivo. Nutlins are non-genotoxic and activate p53 by preventing it from binding to MDM2 (28Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E. Science. 2004; 303: 844-848Crossref PubMed Scopus (3734) Google Scholar). They do not bind to p53 protein and do not interfere with its activities. Treatment of cultured cells with MDM2 antagonists cause accumulation of p53 protein that is free of phosphorylation on Ser15 (28Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E. Science. 2004; 303: 844-848Crossref PubMed Scopus (3734) Google Scholar). Therefore, nutlins may represent valuable molecular tools for studying the role of p53 phosphorylation in its natural cellular context. Here, we show that p53 induced by the MDM2 antagonist, nutlin-3, is not phosphorylated on six key serine residues. Despite the lack of detectable phosphorylation, nutlin-induced p53 showed equal or better sequence-specific DNA binding, ability to transactivate p53 target genes, and p53-dependent apoptotic activity than phosphorylated p53 induced by the genotoxic drugs doxorubicin and etoposide. Our results provide further support to the notion that separating MDM2 from p53 is an important step in p53 activation, but phosphorylation is not required for execution of p53 biological functions.EXPERIMENTAL PROCEDURESCells and Drug Treatment—HCT116 cells were purchased from ATCC (Manassas, VA), and RKO cells were a gift from Dr. B. Vogelstein (Johns Hopkins Oncology Center). Both cell lines have been derived from human colon cancer and possess wild-type p53. Cells were grown in the recommended media supplemented with 10% heat-inactivated fetal bovine serum. Media and serum were purchased from Invitrogen. For drug treatment, 1.5 × 106 cells were seeded in 75-cm2 tissue culture flasks in 10 ml of growth medium 24 h prior to treatment. They were incubated with doxorubicin or etoposide (Sigma, 10 mm stock solution in Me2SO) at various concentrations for 24 h. Control cells were treated with an equivalent amount of Me2SO. RKO-R cells were generated by continuous passage of RKO cells in media containing increasing concentrations of nutlin-3 (0.5–10 μm) over a 90-day period. The resistant cell population was maintained in the presence of 10 μm nutlin-3. p53 gene status was determined by the GeneChip p53 assay (Affymetrix, Santa Clara, CA) as described previously (29Ahrendt S.A. Halachmi S. Chow J.T. Wu L. Halachmi N. Yang S.C. Wehage S. Jen J. Sidransky D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7382-7387Crossref PubMed Scopus (211) Google Scholar).Western Blot Analysis—Cells were harvested by centrifugation and resuspended in lysis buffer containing 20 mm HEPES, 350 mm NaCL, 1 mm MgCL, 0.5 mm EDTA, 0.5 mm dithiothreitol, 20% glycerol, 1% Nonidet P-40, phosphatase inhibitor mixture, and protease inhibitor mixture. Cell pellets were sonicated briefly and cell debris sedimented by brief centrifugation (15,000 rpm) at 4 °C. Supernatants were transferred to fresh tubes, and protein content was determined by the Bradford assay (Bio-Rad). For Western analysis, 10 μg of total protein was loaded onto 4–12% Tris-glycine polyacrylamide gels and subjected to electrophoresis. Proteins were visualized by ECL chemiluminescence reagents (Amersham Biosciences) using primary antibodies specific for human p53 (SC-263, Santa Cruz Biotechnology, Santa Cruz, CA), phospho-p53 (Ser6, Ser15, Ser20, Ser37, Ser46, Ser392; catalog number 9919, Cell Signaling, Beverly MA), p21 (OP64, Oncogene Research Products, Boston, MA), MDM2 (SC-965, Santa Cruz Biotechnology), and β-actin (Sigma). Secondary antibodies used were anti-mouse IgG horseradish peroxidase-linked whole antibody from sheep (NA931V; Amersham Biosciences) and anti-rabbit Ig horseradish peroxidase-linked donkey F(ab′)2 fragment (NA9340V; Amersham Biosciences).p53-DNA Binding Enzyme-linked Immunosorbent Assay—TransAM™ p53 transcription factor assay kit (Active Motif, Carlsbad, CA) was used following manufacturer's protocol. Cell lysates from treated cells were diluted to 2 μg/ml total protein with lysis buffer and applied to plates containing immobilized oligonucleotide containing the p53 consensus binding site (5′-GGACATGCCCGGGCATGTCC-3′). After 1-h incubation at room temperature, plates were washed and incubated with diluted p53 antibody (1:1000) for another hour. Diluted anti-rabbit horseradish peroxidase-conjugated antibody (1:1000) was then added to previously washed plates and developing solution was added and incubated for 8 min to allow color development. The reaction was stopped and absorbance read at 450 nm with a reference wavelength of 650 nm.Quantitative PCR—Cells were seeded in 96-well plates (104 cells/well) 24 h prior to treatment. They were lysed and total RNA extracted using the ABI 6700 robotic work station (Applied Biosystems, Foster City, CA). Aliquots containing 5 μg of total RNA were converted to cDNA using the TaqMan reverse transcription reagents kit (Applied Biosystems). The relative quantity of the p53, p21, and MDM2 transcripts was determined by TaqMan using gene-specific primer/probe sets and 18 S RNA as a normalization control. The sequence of the primers and probes was as follows: p53 (forward, CTG-GGA-CGG-AAC-AGC-TTT-GA; reverse, CCT-TTC-TTG-CGG-AGA-TTC-TCT-TC; probe, CTG-TGC-GCC-GGT-CTC-TCC-CAG-TA), P21 (forward, CTGAGA-CTC-TCA-GGG-TCG-AA; reverse, CGG-CGT-TTG-GAG-TGG-TAG-AA; probe, TTG-GCT-CAC-TGC-AAG-CTC-GCC-CTT), MDM2 (forward, GCT-GGA-GTC-CAG-TGG-GTG-AT; reverse, GAT-GAC-TGT-AGG-CCA-AGC-TAA-TTG; probe, TGG-CTC-ACT-GCA-AGC-TCTGCC-CT), MIC-1 1The abbreviation used is: MIC-1, macrophage inhibitory cytokine-1. (macrophage inhibitory cytokine-1) (forward, CCATGG-TGC-TCA-TTC-AAA-AGA-C; reverse, GGA-AGG-ACC-AGG-ACT-GCT-CAT; probe, TGA-CTT-GTT-AGC-CAA-AGACTG-CCACTG-CA).Apoptosis Assays—Cells were seeded in 24-well tissue culture plates (5 × 104 cells/well) 24 h prior to drug treatment and incubated with the drug for additional 48 h. No treatment controls were established in parallel for each cell line. Culture medium that may contain detached cells was collected and attached cells were trypsinized. Cells were combined with corresponding medium and collected by centrifugation at 1500 rpm for 10 min at 4 °C. Annexin V-positive cells were quantified using Guava Nexin™ kit and the Guava personal cell Analyzer (Guava Technologies, Hayward, CA.) as recommended by the manufacturer.RESULTSMDM2 Antagonists Stabilize p53 and Activate p53 Target Genes—Recently, we developed a class of potent and selective inhibitors of p53-MDM2 interaction (28Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E. Science. 2004; 303: 844-848Crossref PubMed Scopus (3734) Google Scholar). These compounds, called nutlins, bind MDM2 at the p53 pocket with high specificity and can displace p53 from its complex with its negative regulator. Treatment of cells encoding wild-type p53 with nutlins leads to p53 stabilization, accumulation, and activation of the p53 pathway. As nutlins activate p53 by preventing its physical interaction with MDM2, they should not alter the post-translational modification status of p53. Consequently, they represent valuable molecular probes to study the contributions of post-translational modifications to p53 function.Using the MDM2 antagonist nutlin-3, we aimed at studying the functional activity of unmodified p53 compared with the activity of p53 modified in response to treating cells with the genotoxic drugs etoposide and doxorubicin. We chose the colon cancer cell lines HCT116 and RKO as they possess wild-type p53 and respond to genotoxic stress by p53 stabilization and activation of the p53 pathway (28Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E. Science. 2004; 303: 844-848Crossref PubMed Scopus (3734) Google Scholar). To find the optimal treatment condition, we incubated exponentially growing cells with a range of concentrations of etoposide, doxorubicin, and the active enantiomer of nutlin-3 (nutlin-3a) for 24 h. These ranges included the IC50 and IC90 values previously determined by a proliferation/viability assay (28Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E. Science. 2004; 303: 844-848Crossref PubMed Scopus (3734) Google Scholar). Western analysis of the cell lysates from both cells lines revealed a dose-dependent accumulation of p53 and its target gene products MDM2 and p21Waf1/CIP1 (Fig. 1). The observed decrease in the MDM2 and p21 level at the high doxorubicin concentrations is most likely due to protein degradation in cells undergoing apoptosis. This experiment showed that nutlin-3a treatment of HCT116 and RKO cells activates p53 comparably with the genotoxic drugs etoposide and doxorubicin.p53 Induced by MDM2 Antagonists Is Not Modified on Key Phosphorylation Sites—We previously showed that nutlin-1 does not cause p53 phosphorylation at Ser15, a site typically modified in response to genotoxic stress (28Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E. Science. 2004; 303: 844-848Crossref PubMed Scopus (3734) Google Scholar). This observation is consistent with the notion that MDM2 antagonists are non-genotoxic and should not activate the damage-responsive kinases that trigger p53 phosphorylation. However, it is conceivable that other nutlins could possess off-target activities that generate DNA damage or activate stress-related kinases to induce p53 modification. We therefore determined whether nutlin-3a induced p53 phosphorylation on six key serine residues (Ser6, Ser15, Ser20, Ser37, Ser46, and Ser392) using phosphoserine-specific antibodies. The inactive enantiomer nutlin-3b, which has a 150-fold lower affinity to MDM2 in vitro, was used as a negative control (28Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E. Science. 2004; 303: 844-848Crossref PubMed Scopus (3734) Google Scholar).Western analysis showed a comparable accumulation of p53 in both HCT116 and RKO cells treated with etoposide, doxorubicin, and nutlin-3a, but no accumulation was observed with nutlin-3b. Doxorubicin treatment induced the phosphorylation of all examined serine residues of p53 (Fig. 2). Etoposide showed a strong phosphorylation signal on Ser6, Ser15, and Ser20 in both cell lines and Ser46 in RKO cells and weaker but detectable phosphorylation on Ser37 and Ser392. In contrast, phosphorylation of all p53 serines was undetectable in the lysates from both cell lines incubated with nutlin-3a and nutlin-3b. This result confirmed and extended the previous observation made with nutlin-1 that MDM2 antagonists do not induce stress-related modifications previously correlated with p53 activation. Therefore, nutlin-activated p53 provides an opportunity for studying the functional contributions of phosphorylation to p53 function in living cells.Fig. 2Stabilization of p53 by MDM2 antagonists does not involve phosphorylation of key serine residues. Exponentially growing HCT116 and RKO cells were incubated with etoposide (10 μm), doxorubicin (1 μm), nutlin-3a (10 μm), or nutlin-3b (10 μm) for 24 h, and the levels of total p53 and p53 phosphorylated on specific serine residues were analyzed by Western blotting. Actin was used as a normalization control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DNA Binding Activity of p53 Does Not Depend on Its Phosphorylation Status—p53 transcriptional activity is of paramount importance for its function as a tumor suppressor. We therefore evaluated the ability of p53 to bind its DNA recognition sequences as an indication of the transcriptional activation potential of the transcription factor. We used the TransAM™ p53 enzyme-linked immunosorbent assay that measures the relative amount of p53 in cell lysates that can bind to a 20-mer oliginucleotide containing a p53 consensus binding site. HCT116 and RKO cells were incubated with a range of concentrations of etoposide, doxorubicin, and nutlin-3a for 24 h, and DNA-bound p53 was assayed in the cell lysates (Fig. 3). In agreement with the Western analyses (Fig. 1), the levels of DNA-bound p53 increased in a dose-dependent manner in both cell lines treated with all three drugs. The 14–17-fold elevation of p53 in RKO cells was more dramatic, while HCT116 cells showed a more moderate (5–6-fold) increase that reflects the higher basal level of p53 in these cells. The level of p53 induced by nutlin-3a was higher than the level induced by either etoposide or doxorubicin in HCT116 cells and comparable with that induced by these drugs (14-fold versus 16–17-fold) in RKO cells. These data suggest that the lack of detectable phosphorylation on six major phosphorylation sites does not affect the ability of p53 to bind effectively its DNA response elements.Fig. 3Binding of p53 to its consensus DNA sequence is not affected by its phosphorylation status in vivo. HCT116 and RKO cells were incubated with doxorubicin, etoposide, and nutlins-3a for 24 h, and the level of p53 protein present in the cell lysates that can bind to its consensus recognition sequence was determined by the TransAM™ p53 enzyme-linked immunosorbent assay and calculated as fold increase relative to the control samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Transcriptional Activity of p53 Is Not Affected by Its Phosphorylation Status—We next compared the transcriptional activities of phosphorylated and unphosphorylated p53 after treatment of HCT116 and RKO cells with increasing concentrations of etoposide, doxorubicin, nutlin-3a, and nutlin-3b for 24 h. We measured the expression of three p53 target genes (p21Waf1, mdm2, and mic-1) by quantitative real-time PCR. These genes contain p53 recognition sequences in their promoter regions, strongly depend on p53 for transcriptional regulation, and represent diverse functions of the p53 pathway: p21Waf1/CIP1 encodes a potent cyclin-dependent kinase inhibitor that plays a key role in the p53-mediated cell cycle arrest (30el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7890) Google Scholar); MDM2 is a p53 negative regulator (31Picksley S.M. Lane D.P. Bioessays. 1993; 15: 689-690Crossref PubMed Scopus (172) Google Scholar); and the recently discovered transforming growth factor-β superfamily member, MIC-1, is a secreted protein with poorly understood function (32Tan M. Wang Y. Guan K. Sun Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 109-114Crossref PubMed Scopus (227) Google Scholar, 33Yang H. Filipovic Z. Brown D. Breit S.N. Vassilev L.T. Mol. Cancer Ther. 2003; 2: 1023-1029PubMed Google Scholar).Dose-dependent accumulation of p53 in drug-treated cells elicited a dose-dependent activation of all three genes in both cell lines (Fig. 4A). The relative increase in gene expression was different in each cell line, probably reflecting differences in the basal level of expression. p21 was most highly elevated in HCT116, while MIC-1 showed the strongest induction in RKO cells. To compare the transcriptional activity of p53 between drug-treated cells the highest level of induction within the concentration range was plotted for each drug and cell line (Fig. 4B). The level of gene induction by nutlin-3a (10–12-fold) was the highest for all three genes in HCT116 cells and for p21 and MDM2 in RKO cells. Only the expression of the MIC-1 gene was slightly higher in doxorubicin-treated RKO cells (Fig. 4B). The inactive enantiomer (nutlin-3b) did not show significant transcriptional activation of any of the genes in either cell line, confirming that the activation of p53 target genes by nutlin-3a is due to inhibition of MDM2-p53 interaction (28Vassilev L.T. Vu B.T. Graves B. Carvajal D. Podlaski F. Filipovic Z. Kong N. Kammlott U. Lukacs C. Klein C. Fotouhi N. Liu E. Science. 2004; 303: 844-848Crossref PubMed Scopus (3734) Google Scholar).Fig. 4Activation of p53-regulated genes in cancer cells does not depend on the phosphorylation status of p53. Exponentially growing HCT116 and RKO cells were incubated with the indicated concentration of doxorubicin (Dox), etoposide (Etopo), nutlin-3a, and nutlin-3b for 24 h, and the relative expression of three p53-regulated genes (p21, mdm2, and mic-1) was determined by quantitative PCR. They were plotted as a relative increase in gene activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression of p53 target genes was measured after 24 h of drug treatment to reach a steady-state level of p53 and to avoid possible differences in the timing of p53 induction. However, one could argue that p53 phosphorylation accelerates p53 activation by both antagonizing MDM2 interaction and enabling recruitment of co-activators. On the other hand, if the critical step in p53 activation involves preventing MDM2 binding, then nutlin-3a may provide a more direct and rapid route to activation, since kinase activation, p53 modification, and damage-induced degradation of MDM2 (6Stommel J.M. Wahl G.M. EMBO J. 2004; 23: 1547-1556Crossref PubMed Scopus (312) Google Scholar) w