Identification of New p53 Acetylation Sites in COS-1 Cells
2009; Elsevier BV; Volume: 8; Issue: 6 Linguagem: Inglês
10.1074/mcp.m800487-mcp200
ISSN1535-9484
AutoresAnita Joubel, Robert J. Chalkley, Katalin F. Medzihradszky, Hubert Hondermarck, Alma L. Burlingame,
Tópico(s)Epigenetics and DNA Methylation
ResumoThe p53 tumor suppressor protein is a key regulator of cell cycle and death that is involved in many cell signaling pathways and is tightly regulated in mammalian cells. Post-translational modifications of p53 have been investigated previously mainly using antibodies. In this study, utilizing LC-MS/MS analysis, we have characterized p53 protein from COS-1 cells. Several already known post-translational modifications were observed, such as phosphorylation on serines 15, 33, 315, and 392 as well as acetylation on lysines 305, 370, 372, 373, 381, 382, and 386. Interestingly novel acetylation sites were identified at lysines 319 and 357. This study confirmed that p53 is a highly acetylated protein and revealed new acetylation sites that might aid the further understanding of p53 regulation. The p53 tumor suppressor protein is a key regulator of cell cycle and death that is involved in many cell signaling pathways and is tightly regulated in mammalian cells. Post-translational modifications of p53 have been investigated previously mainly using antibodies. In this study, utilizing LC-MS/MS analysis, we have characterized p53 protein from COS-1 cells. Several already known post-translational modifications were observed, such as phosphorylation on serines 15, 33, 315, and 392 as well as acetylation on lysines 305, 370, 372, 373, 381, 382, and 386. Interestingly novel acetylation sites were identified at lysines 319 and 357. This study confirmed that p53 is a highly acetylated protein and revealed new acetylation sites that might aid the further understanding of p53 regulation. p53 plays a key role in cellular homeostasis and is at the heart of a complex network of protective mechanisms safeguarding cellular integrity. Because of its central function in processes such as cell cycle regulation, apoptosis, DNA repair, cellular senescence, and apoptosis, the p53 pathway is crucial for effective tumor suppression, and mutations in p53 that compromise its function occur in more than 50% of cancers (1Aylon Y. Oren M. Living with p53, dying of p53.Cell. 2007; 130: 597-600Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 2Van Dyke T. Cancer biology: sense out of missense.Nature. 2005; 434: 287-288Crossref PubMed Scopus (8) Google Scholar). Interestingly p53 appears to be highly post-translationally modified, and although ubiquitination, neddylation, sumoylation, and methylation have been described, phosphorylation and acetylation are the most commonly reported modifications of p53 (3Xu Y. Regulation of p53 responses by post-translational modifications.Cell Death Differ. 2003; 10: 400-403Crossref PubMed Scopus (244) Google Scholar). Both phosphorylation and acetylation affect p53 stability and activity and are induced following various types of stress (4Appella E. Anderson C.W. Post-translational modifications and activation of p53 by genotoxic stresses.Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (902) Google Scholar). For example, phosphorylation at Ser15, Ser20, Thr18, and Ser37 disrupts the interaction between p53 and its major negative regulator, MDM2 (3Xu Y. Regulation of p53 responses by post-translational modifications.Cell Death Differ. 2003; 10: 400-403Crossref PubMed Scopus (244) Google Scholar, 5Jabbur J.R. Tabor A.D. Cheng X. Wang H. Uesugi M. Lozano G. Zhang W. Mdm-2 binding and TAF(II)31 recruitment is regulated by hydrogen bond disruption between the p53 residues Thr18 and Asp21.Oncogene. 2002; 21: 7100-7113Crossref PubMed Scopus (38) Google Scholar), leading to an increase of p53 protein expression and activity. The acetylation sites are located mostly in the C-terminal end of p53 where the tetramerization and regulatory domains localize. Sites of acetylation have been reported at lysine residues 120, 164, 305, 320, 370, 372, 373, 381, 382, and 386 (6Gu W. Roeder R.G. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain.Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar, 7Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. DNA damage activates p53 through a phosphorylation-acetylation cascade.Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1013) Google Scholar, 8Tang Y. Luo J. Zhang W. Gu W. Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis.Mol. Cell. 2006; 24: 827-839Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 9Wang Y.H. Tsay Y.G. Tan B.C. Lo W.Y. Lee S.C. Identification and characterization of a novel p300-mediated p53 acetylation site, lysine 305.J. Biol. Chem. 2003; 278: 25568-25576Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 10Ivanov G.S. Ivanova T. Kurash J. Ivanov A. Chuikov S. Gizatullin F. Herrera-Medina E.M. Rauscher III, F. Reinberg D. Barlev N.A. Methylation-acetylation interplay activates p53 in response to DNA damage.Mol. Cell. Biol. 2007; 27: 6756-6769Crossref PubMed Scopus (145) Google Scholar, 11Li A.G. Piluso L.G. Cai X. Gadd B.J. Ladurner A.G. Liu X. An acetylation switch in p53 mediates holo-TFIID recruitment.Mol. Cell. 2007; 28: 408-421Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 12Kurash J.K. Lei H. Shen Q. Marston W.L. Granda B.W. Fan H. Wall D. Li E. Gaudet F. Methylation of p53 by Set7/9 mediates p53 acetylation and activity in vivo.Mol. Cell. 2008; 29: 392-400Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 13Barlev N.A. Liu L. Chehab N.H. Mansfield K. Harris K.G. Halazonetis T.D. Berger S.L. Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases.Mol. Cell. 2001; 8: 1243-1254Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 14Tang Y. Zhao W. Chen Y. Zhao Y. Gu W. Acetylation is indispensable for p53 activation.Cell. 2008; 133: 612-626Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar), and importantly, acetylation has recently been shown to be indispensable for p53 activation (14Tang Y. Zhao W. Chen Y. Zhao Y. Gu W. Acetylation is indispensable for p53 activation.Cell. 2008; 133: 612-626Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). In this context of high regulation of p53 through post-translational modifications, we aimed at identifying potential new p53 modifications by using mass spectrometry. p53 was obtained from the kidney fibroblast-like COS-1 cells that are known to produce a high amount of p53. In fact, in these cells, p53 is bound to SV40 large T antigen (15Lane D.P. Crawford L.V. T antigen is bound to a host protein in SV40-transformed cells.Nature. 1979; 278: 261-263Crossref PubMed Scopus (1743) Google Scholar, 16Linzer D.I. Levine A.J. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.Cell. 1979; 17: 43-52Abstract Full Text PDF PubMed Scopus (1232) Google Scholar, 17Reich N.C. Oren M. Levine A.J. Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.Mol. Cell. Biol. 1983; 3: 2143-2150Crossref PubMed Scopus (256) Google Scholar). This association sequesters the gene transactivation function of p53, rendering it inactive as a transcription factor. The sequestration leads to an accumulation of p53 as part of a complex with SV40 large T antigen (17Reich N.C. Oren M. Levine A.J. Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.Mol. Cell. Biol. 1983; 3: 2143-2150Crossref PubMed Scopus (256) Google Scholar). Utilizing CID analysis and high accuracy mass measurements, a number of different modifications, both known and novel, were deciphered. They encompass phosphorylation of serine residues 15, 33, 315, and 392 and acetylation of lysines 305, 319, 357, 370, 372, 373, 381, 382, and 386. The acetylation of p53 at Lys319 and Lys357 is reported for the first time.DISCUSSIONWe have identified phosphorylation and acetylation sites on endogenous p53 from COS-1 cells. The phosphorylation sites detected were localized at the N- and C-terminal ends of the protein on serines 15, 33, 315, and 392. Our results confirmed some phosphorylation sites previously described by Tack and Wright (20Tack L.C. Wright J.H. Altered phosphorylation of free and bound forms of monkey p53 and simian virus 40 large T antigen during lytic infection.J. Virol. 1992; 66: 1312-1320Crossref PubMed Google Scholar) using Edman sequencing and radiolabeling. They identified phosphorylation on Ser9, Ser15, Ser20, either Ser33 or Ser37, at least one of Ser90 or Ser99, Ser315, and Ser392. The new results presented here were able to pinpoint phosphorylation on Ser33, resolving ambiguity for one of the sites in their study. The additional phosphorylations described by Tack and Wright (20Tack L.C. Wright J.H. Altered phosphorylation of free and bound forms of monkey p53 and simian virus 40 large T antigen during lytic infection.J. Virol. 1992; 66: 1312-1320Crossref PubMed Google Scholar) that were not detected in the current study might be related to the use by the authors of CV-1 cells, the parent cell line of the COS-1 cells used in our present study. They infected the CV-1 cells with SV40 wild-type virus. However, the COS-1 cell line is stable because it contains only a defective mutant of SV40 that codes only for wild-type T antigen. The new discoveries presented here are the acetylation sites in the C-terminal region of p53. Acetylation occurred on Lys residues in positions 305, 319, 357, 370, 372, 373, 381, 382, and 386. None of these sites have been described previously on endogenous p53 from COS-1 cells, although many have been reported in other species (4Appella E. Anderson C.W. Post-translational modifications and activation of p53 by genotoxic stresses.Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (902) Google Scholar, 21Blaydes J.P. Luciani M.G. Pospisilova S. Ball H.M. Vojtesek B. Hupp T.R. Stoichiometric phosphorylation of human p53 at Ser315 stimulates p53-dependent transcription.J. Biol. Chem. 2001; 276: 4699-4708Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 22Lavin M.F. Gueven N. The complexity of p53 stabilization and activation.Cell Death Differ. 2006; 13: 941-950Crossref PubMed Scopus (529) Google Scholar). Work by Borger and DeCaprio (25Borger D.R. DeCaprio J.A. Targeting of p300/CREB binding protein coactivators by simian virus 40 is mediated through p53.J. Virol. 2006; 80: 4292-4303Crossref PubMed Scopus (31) Google Scholar) described the acetylation of SV40 large T-bound p53 at lysine 373 by Western blot analysis. In their experiment, they used p53 co-immunoprecipitated with SV40 large T from whole-cell lysates obtained from human osteosarcoma U-2 OS cells stably expressing wild-type SV40 large T antigen.Importantly two acetylation sites identified in our study have never been described so far and are located on lysines 319 and 357. Although mutations at lysine 319 have occasionally been detected in non-small cell lung carcinomas (23Gorgoulis V.G. Zacharatos P. Kotsinas A. Liloglou T. Kyroudi A. Veslemes M. Rassidakis A. Halazonetis T.D. Field J.K. Kittas C. Alterations of the p16-pRb pathway and the chromosome locus 9p21–22 in non-small-cell lung carcinomas: relationship with p53 and MDM2 protein expression.Am. J. Pathol. 1998; 153: 1749-1765Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar) and hepatocellular carcinomas (24Teramoto T. Satonaka K. Kitazawa S. Fujimori T. Hayashi K. Maeda S. p53 gene abnormalities are closely related to hepatoviral infections and occur at a late stage of hepatocarcinogenesis.Cancer Res. 1994; 54: 231-235PubMed Google Scholar), lysines 319 and 357 have never been reported to be post-translational modification sites, and consequently, their potential roles in p53 regulation have not been explored. Our results provide novel and interesting observations as they were obtained without stressing the cells. Modifications on p53 are triggered by stress. Few residues are thought to be constitutively modified. Constitutive phosphorylations at Ser6, Ser33, Ser315, and Ser392 (26Saito S. Yamaguchi H. Higashimoto Y. Chao C. Xu Y. Fornace Jr., A.J. Appella E. Anderson C.W. Phosphorylation site interdependence of human p53 post-translational modifications in response to stress.J. Biol. Chem. 2003; 278: 37536-37544Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar) as well as at Ser376 or Ser378 (27Waterman M.J. Stavridi E.S. Waterman J.L. Halazonetis T.D. ATM-dependent activation of p53 involves dephosphorylation and association with 14-3-3 proteins.Nat. Genet. 1998; 19: 175-178Crossref PubMed Scopus (402) Google Scholar) have been reported. Three of these modifications (Ser33, Ser315, and Ser392) were observed in our experiments. The role of these modifications has not been clearly established. It has been demonstrated that, in vitro, phosphorylation of Ser392 stimulated formation of p53 tetramers, whereas phosphorylation of Ser315 reversed it (28Sakaguchi K. Sakamoto H. Xie D. Erickson J.W. Lewis M.S. Anderson C.W. Appella E. Effect of phosphorylation on tetramerization of the tumor suppressor protein p53.J. Protein Chem. 1997; 16: 553-556Crossref PubMed Scopus (33) Google Scholar). p53 binds to large T antigen as a monomer (29Lilyestrom W. Klein M.G. Zhang R. Joachimiak A. Chen X.S. Crystal structure of SV40 large T-antigen bound to p53: interplay between a viral oncoprotein and a cellular tumor suppressor.Genes Dev. 2006; 20: 2373-2382Crossref PubMed Scopus (109) Google Scholar). Does a balance between the two modifications lead to a tendency toward a p53 monomer formation in COS-1 cells? Ser15 phosphorylation along with Thr18, Ser20, and Ser37 has been shown to lead to a conformational change in p53 that prevents its interaction with MDM2, thus inhibiting p53 ubiquitination and degradation. In our case, only Ser15 was detected as phosphorylated. We can hypothesize that the binding of large T antigen to p53 by itself could stabilize p53. Hence it has been proposed that large T antigen enhances the stability of p53 partly by complexing with MDM2 (30Brown D.R. Deb S. Munoz R.M. Subler M.A. Deb S.P. The tumor suppressor p53 and the oncoprotein simian virus 40 T antigen bind to overlapping domains on the MDM2 protein.Mol. Cell. Biol. 1993; 13: 6849-6857Crossref PubMed Scopus (56) Google Scholar, 31Henning W. Rohaly G. Kolzau T. Knippschild U. Maacke H. Deppert W. MDM2 is a target of simian virus 40 in cellular transformation and during lytic infection.J. Virol. 1997; 71: 7609-7618Crossref PubMed Google Scholar). In our experiment, we identified large T antigen in the p53 pulldown assay but not MDM2. p53 N-terminal phosphorylation can also promote the binding of the acetyltransferases CBP 1The abbreviation used is:CBPcAMP-response element-binding protein (CREB)-binding protein./p300 to p53 (3Xu Y. Regulation of p53 responses by post-translational modifications.Cell Death Differ. 2003; 10: 400-403Crossref PubMed Scopus (244) Google Scholar). Kishi et al. (32Kishi H. Nakagawa K. Matsumoto M. Suga M. Ando M. Taya Y. Yamaizumi M. Osmotic shock induces G1 arrest through p53 phosphorylation at Ser33 by activated p38MAPK without phosphorylation at Ser15 and Ser20.J. Biol. Chem. 2001; 276: 39115-39122Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) suggested that phosphorylation of Ser33 may stimulate acetylation of p53. Upon stress, p53 is known to be specifically acetylated at lysine residues (in positions 164, 370, 372, 373, 381, and 382) by CBP/p300 (6Gu W. Roeder R.G. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain.Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar, 13Barlev N.A. Liu L. Chehab N.H. Mansfield K. Harris K.G. Halazonetis T.D. Berger S.L. Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases.Mol. Cell. 2001; 8: 1243-1254Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar), at Lys320 by p300/CBP-associated factor (PCAF) (7Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. DNA damage activates p53 through a phosphorylation-acetylation cascade.Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1013) Google Scholar), at Lys305 by p300 (10Ivanov G.S. Ivanova T. Kurash J. Ivanov A. Chuikov S. Gizatullin F. Herrera-Medina E.M. Rauscher III, F. Reinberg D. Barlev N.A. Methylation-acetylation interplay activates p53 in response to DNA damage.Mol. Cell. Biol. 2007; 27: 6756-6769Crossref PubMed Scopus (145) Google Scholar), and at Lys120 by the p19ARF/oncogene pathway (33Sykes S.M. Mellert H.S. Holbert M.A. Li K. Marmorstein R. Lane W.S. McMahon S.B. Acetylation of the p53 DNA-binding domain regulates apoptosis induction.Mol. Cell. 2006; 24: 841-851Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar, 34Mellert H. Sykes S.M. Murphy M.E. McMahon S.B. The ARF/oncogene pathway activates p53 acetylation within the DNA binding domain.Cell Cycle. 2007; 6: 1304-1306Crossref PubMed Scopus (22) Google Scholar). It was also demonstrated that Tip60, Ada3, and Pin1 participate in the control of p53 acetylation (8Tang Y. Luo J. Zhang W. Gu W. Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis.Mol. Cell. 2006; 24: 827-839Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 35Nag A. Germaniuk-Kurowska A. Dimri M. Sassack M.A. Gurumurthy C.B. Gao Q. Dimri G. Band H. Band V. An essential role of human Ada3 in p53 acetylation.J. Biol. Chem. 2007; 282: 8812-8820Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 36Mantovani F. Tocco F. Girardini J. Smith P. Gasco M. Lu X. Crook T. Del Sal G. The prolyl isomerase Pin1 orchestrates p53 acetylation and dissociation from the apoptosis inhibitor iASPP.Nat. Struct. Mol. Biol. 2007; 14: 912-920Crossref PubMed Scopus (129) Google Scholar). The effects of acetylation on p53 function are not totally clear, but they have been implicated in modulating p53 transcriptional activity and stability (37Prives C. Manley J.L. Why is p53 acetylated?.Cell. 2001; 107: 815-818Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Like the acetylation of lysine residues in histones, acetylation of p53 has been linked to gene transcription regulation. Knights et al. (38Knights C.D. Catania J. Di Giovanni S. Muratoglu S. Perez R. Swartzbeck A. Quong A.A. Zhang X. Beerman T. Pestell R.G. Avantaggiati M.L. Distinct p53 acetylation cassettes differentially influence gene-expression patterns and cell fate.J. Cell Biol. 2006; 173: 533-544Crossref PubMed Scopus (209) Google Scholar) showed that distinct p53 acetylation “cassettes” differentially influence gene expression patterns and cell survival versus death. Acetylation sites are also important to keep p53 from ubiquitination and subsequent degradation by MDM2 as p53 is ubiquitinated and acetylated on similar sites at the C terminus (39Ito A. Lai C.H. Zhao X. Saito S. Hamilton M.H. Appella E. Yao T.P. p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2.EMBO J. 2001; 20: 1331-1340Crossref PubMed Scopus (435) Google Scholar, 40Rodriguez M.S. Desterro J.M. Lain S. Lane D.P. Hay R.T. Multiple C-terminal lysine residues target p53 for ubiquitin-proteasome-mediated degradation.Mol. Cell. Biol. 2000; 20: 8458-8467Crossref PubMed Scopus (300) Google Scholar), suggesting that these modifications may compete for the same residues. Importantly recent work by Tang et al. (14Tang Y. Zhao W. Chen Y. Zhao Y. Gu W. Acetylation is indispensable for p53 activation.Cell. 2008; 133: 612-626Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar) described p53 acetylation as an indispensable event that destabilizes the p53-MDM2 interaction enabling p53 activation. In our study, lysines 319 and 357, which were observed to be acetylated, border the tetramerization domain of p53 (Fig. 4). The recent crystal structure of large T antigen complexed with p53 revealed a hexameric complex of large T antigen binding six p53 monomers (29Lilyestrom W. Klein M.G. Zhang R. Joachimiak A. Chen X.S. Crystal structure of SV40 large T-antigen bound to p53: interplay between a viral oncoprotein and a cellular tumor suppressor.Genes Dev. 2006; 20: 2373-2382Crossref PubMed Scopus (109) Google Scholar), and one could speculate that the new acetylation sites that we report here might impact p53 conformation and eventually participate in the formation and stabilization of the complex with large T antigen.In conclusion, our study confirmed that p53 is a highly acetylated protein and unveiled new acetylation sites. Given the increasingly reported importance of acetylation for the regulation of p53, and although further functional exploration will be needed, the new acetylation sites identified here open new perspectives for the refinement of p53 mechanism of regulation. p53 plays a key role in cellular homeostasis and is at the heart of a complex network of protective mechanisms safeguarding cellular integrity. Because of its central function in processes such as cell cycle regulation, apoptosis, DNA repair, cellular senescence, and apoptosis, the p53 pathway is crucial for effective tumor suppression, and mutations in p53 that compromise its function occur in more than 50% of cancers (1Aylon Y. Oren M. Living with p53, dying of p53.Cell. 2007; 130: 597-600Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 2Van Dyke T. Cancer biology: sense out of missense.Nature. 2005; 434: 287-288Crossref PubMed Scopus (8) Google Scholar). Interestingly p53 appears to be highly post-translationally modified, and although ubiquitination, neddylation, sumoylation, and methylation have been described, phosphorylation and acetylation are the most commonly reported modifications of p53 (3Xu Y. Regulation of p53 responses by post-translational modifications.Cell Death Differ. 2003; 10: 400-403Crossref PubMed Scopus (244) Google Scholar). Both phosphorylation and acetylation affect p53 stability and activity and are induced following various types of stress (4Appella E. Anderson C.W. Post-translational modifications and activation of p53 by genotoxic stresses.Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (902) Google Scholar). For example, phosphorylation at Ser15, Ser20, Thr18, and Ser37 disrupts the interaction between p53 and its major negative regulator, MDM2 (3Xu Y. Regulation of p53 responses by post-translational modifications.Cell Death Differ. 2003; 10: 400-403Crossref PubMed Scopus (244) Google Scholar, 5Jabbur J.R. Tabor A.D. Cheng X. Wang H. Uesugi M. Lozano G. Zhang W. Mdm-2 binding and TAF(II)31 recruitment is regulated by hydrogen bond disruption between the p53 residues Thr18 and Asp21.Oncogene. 2002; 21: 7100-7113Crossref PubMed Scopus (38) Google Scholar), leading to an increase of p53 protein expression and activity. The acetylation sites are located mostly in the C-terminal end of p53 where the tetramerization and regulatory domains localize. Sites of acetylation have been reported at lysine residues 120, 164, 305, 320, 370, 372, 373, 381, 382, and 386 (6Gu W. Roeder R.G. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain.Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2152) Google Scholar, 7Sakaguchi K. Herrera J.E. Saito S. Miki T. Bustin M. Vassilev A. Anderson C.W. Appella E. DNA damage activates p53 through a phosphorylation-acetylation cascade.Genes Dev. 1998; 12: 2831-2841Crossref PubMed Scopus (1013) Google Scholar, 8Tang Y. Luo J. Zhang W. Gu W. Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis.Mol. Cell. 2006; 24: 827-839Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 9Wang Y.H. Tsay Y.G. Tan B.C. Lo W.Y. Lee S.C. Identification and characterization of a novel p300-mediated p53 acetylation site, lysine 305.J. Biol. Chem. 2003; 278: 25568-25576Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 10Ivanov G.S. Ivanova T. Kurash J. Ivanov A. Chuikov S. Gizatullin F. Herrera-Medina E.M. Rauscher III, F. Reinberg D. Barlev N.A. Methylation-acetylation interplay activates p53 in response to DNA damage.Mol. Cell. Biol. 2007; 27: 6756-6769Crossref PubMed Scopus (145) Google Scholar, 11Li A.G. Piluso L.G. Cai X. Gadd B.J. Ladurner A.G. Liu X. An acetylation switch in p53 mediates holo-TFIID recruitment.Mol. Cell. 2007; 28: 408-421Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 12Kurash J.K. Lei H. Shen Q. Marston W.L. Granda B.W. Fan H. Wall D. Li E. Gaudet F. Methylation of p53 by Set7/9 mediates p53 acetylation and activity in vivo.Mol. Cell. 2008; 29: 392-400Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 13Barlev N.A. Liu L. Chehab N.H. Mansfield K. Harris K.G. Halazonetis T.D. Berger S.L. Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases.Mol. Cell. 2001; 8: 1243-1254Abstract Full Text Full Text PDF PubMed Scopus (583) Google Scholar, 14Tang Y. Zhao W. Chen Y. Zhao Y. Gu W. Acetylation is indispensable for p53 activation.Cell. 2008; 133: 612-626Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar), and importantly, acetylation has recently been shown to be indispensable for p53 activation (14Tang Y. Zhao W. Chen Y. Zhao Y. Gu W. Acetylation is indispensable for p53 activation.Cell. 2008; 133: 612-626Abstract Full Text Full Text PDF PubMed Scopus (642) Google Scholar). In this context of high regulation of p53 through post-translational modifications, we aimed at identifying potential new p53 modifications by using mass spectrometry. p53 was obtained from the kidney fibroblast-like COS-1 cells that are known to produce a high amount of p53. In fact, in these cells, p53 is bound to SV40 large T antigen (15Lane D.P. Crawford L.V. T antigen is bound to a host protein in SV40-transformed cells.Nature. 1979; 278: 261-263Crossref PubMed Scopus (1743) Google Scholar, 16Linzer D.I. Levine A.J. Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.Cell. 1979; 17: 43-52Abstract Full Text PDF PubMed Scopus (1232) Google Scholar, 17Reich N.C. Oren M. Levine A.J. Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.Mol. Cell. Biol. 1983; 3: 2143-2150Crossref PubMed Scopus (256) Google Scholar). This association sequesters the gene transactivation function of p53, rendering it inactive as a transcription factor. The sequestration leads to an accumulation of p53 as part of a complex with SV40 large T antigen (17Reich N.C. Oren M. Levine A.J. Two distinct mechanisms regulate the levels of a cellular tumor antigen, p53.Mol. Cell. Biol. 1983; 3: 2143-2150Crossref PubMed Scopus (256) Google Scholar). Utilizing CID analysis and high accuracy mass measurements, a number of different modifications, both known and novel, were deciphered. They encompass phosphorylation of serine residues 15, 33, 315, and 392 and acetylation of lysines 305, 319, 357, 370, 372, 373, 381, 382, and 386. The acetylation of p53 at Lys319 and Lys357 is reported for the first time. DISCUSSIONWe have identified phosphorylation and acetylation sites on endogenous p53 from COS-1 cells. The phosphorylation sites detected were localized at the N- and C-terminal ends of the protein on serines 15, 33, 315, and 392. Our results confirmed some phosphorylation sites previously described by Tack and Wright (20Tack L.C. Wright J.H. Altered phosphorylation of free and bound forms of monkey p53 and simian virus 40 large T antigen during lytic infection.J. Virol. 1992; 66: 1312-1320Crossref PubMed Google Scholar) using Edman sequencing and radiolabeling. They identified phosphorylation on Ser9, Ser15, Ser20, either Ser33 or Ser37, at least one of Ser90 or Ser99, Ser315, and Ser392. The new results presented here were able to pinpoint phosphorylation on Ser33, resolving ambiguity for one of the sites in their study. The additional phosphorylations described by Tack and Wright (20Tack L.C. Wright J.H. Altered phosphorylation of free and bound forms of monkey p53 and simian virus 40 large T antigen during lytic infection.J. Virol. 1992; 66: 1312-1320Crossref PubMed Google Scholar) that were not detected in the current study might be related to the use by the authors of CV-1 cells, the parent cell line of the COS-1 cells used in our present study. They infected the CV-1 cells with SV40 wild-type virus. However, the COS-1 cell line is stable because it contains only a defective mutant of SV40 that codes only for wild-type T antigen. The new discoveries presented here are the acetylation sites in the C-terminal region of p53. Acetylation occurred on Lys residues in positions 305, 319, 357, 370, 372, 373, 381, 382, and 386. None of these sites have been described previously on endogenous p53 from COS-1 cells, although many have been reported in other species (4Appella E. Anderson C.W. Post-translational modifications and activation of p53 by genotoxic stresses.Eur. J. Biochem. 2001; 268: 2764-2772Crossref PubMed Scopus (902) Google Scholar, 21Blaydes J.P. Luciani M.G. Pospisilova S. Ball H.M. Vojtesek B. Hupp T.R. Stoichiometric phosphorylation of human p53 at Ser315 stimulates p53-dependent transcription.J. Biol. Chem. 2001; 276: 4699-4708Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 22Lavin M.F. Gueven N. The complexity of p53 stabilization and activation.Cell Death Differ. 2006; 13: 941-950Crossref PubMed Scopus (529) Google Scholar). Work by Borger and DeCapri
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