Animal cells and systems

The Korean Society for Integrative Biology (한국통합생물학회)
권호 표지

DNA Damage-inducible Phosphorylation of p53 at Ser20 is Required for p53 Stabilization

  • Yang, Dong-Hwa (Department of Life Science, College of Natural Sciences, Sogang University) ;
  • Rhee, Byung-Kirl (Department of Life Science, College of Natural Sciences, Sogang University) ;
  • Yim, Tae-Hee (Department of Life Science, College of Natural Sciences, Sogang University) ;
  • Lee, Hye-Jin (Department of Life Science, College of Natural Sciences, Sogang University) ;
  • Kim, Jungho (Department of Life Science, College of Natural Sciences, Sogang University)
  • Published : 2002.09.01
Download PDF
⟨ Previous Next ⟩

Abstract

The p53 tumor suppressor gene is among the most frequently mutated and studied genes in human cancer, but the mechanisms by which it sur presses tumor formation remain unclear. DNA damage regulates both the protein levels of p53 and its affinity for specific DNA sequences. Stabilization of p53 in response to DNA damage is caused by its dissociation from Mdm2, a downstream target gene of p53 and a protein that targets p53 for degradation in the proteosome. Recent studies have suggested that phosphorylation of human p53 at Ser20 is important for stabilizing p53 in response to DNA damage through disruption of the interaction between Mdm2 and p53. We generated mice with an allele encoding changes at Ser20, known to be essential for p53 accumulation following DNA damage, to enable analyses of p53 stabilization in vivo. Our data showed that the mutant p53 was clearly defective for full stabilization of p53 in response to DNA damage. We concluded that Ser20 phosphorylation is critical for modulating the negative regulation of p53 by Mdm2, probably through phosphorylation-dependent inhibition of p53-Mdm2 interaction in the physiological context.

Keywords

References

  1. Ahn JY, Schwart JK, Piwnica-Worms H, and Canman E (2000) Threonine 68 phosphorylation by Ataxia telangiectasia mutated is required for efficient activation of Chk2 in response to ionizing radiation, Cancer Res 60: 5934-5936
  2. Argos P, Landy A, Abremski K, Egan JB, Ljungquist EH, Hoess RH, Kahn ML, Kalionis B, Narayana SVL, Pierson LS, Sternberg N and Leong JM (1986) The intergrase family of site-specific recombinases: regional similarities and global diversity, EMBO J 5: 433-440
  3. Ashcroft M, Kubbutat MH and Vousden KH (1999) Regulation of p53 function and stability by phosphorylation, Mol Cell Biol 19: 1751-1758
  4. Attardi LD, Reczek EE, Cosmas C, Demicco EG, McCurrach ME, Lowe SW, and Jacks T (2000) PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family, Genes Dev 14: 704-718
  5. Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L, Smorodinsky NI, Prives C, Reiss Y, Shiloh Y, and Ziv Y (1998) Enhanced phosphorylation of p53 by ATM in the response to DNA damage, Science 281: 1674-1677 https://doi.org/10.1126/science.281.5383.1674
  6. Bell DW, Varley JM, Szydlo TE, Kang DH, Waher DC, Shannon KE, Lubratovich M, Verselis SJ, Isselbacher KJ, Fraumeni JF, Birch JM, Li FP, Garber JE and Haber DA (1999) Heterozygous germ line hCHK2 mutations in Li-Fraumeni syndrome, Science 286: 2528-2531 https://doi.org/10.1126/science.286.5449.2528
  7. Bienz B ,Zakut-Houri R, Givol D and Oren M (1984) Analysis of the gene coding for the murine cellular antigen p53, EMBO J 3: 79-83
  8. Blaydes JP and Hupp T (1998) DNA damage triggers DRB-resistant phosphorylation of human p53 at the CK2 site, Oncogene 17: 1045-4052 https://doi.org/10.1038/sj.onc.1202014
  9. Blasina A, de Weyer IV, Laus MC, Luyten WH, Parker AF and McGowan CH (1999) A human homologue of the checkpoint kinase Cds 1 directly inhibits Cdc25 phosphatase, Curr Biol 9: 1-10 https://doi.org/10.1016/S0960-9822(99)80041-4
  10. Blatter C, Tobiasch E, Litfen M, Rahmsdorf HJ and Herrlich P (1999) DNA damage induced p53 stabilization: no indication for an involvement of p53 phosphorylation, Oncogene 18: 1723-1732 https://doi.org/10.1038/sj.onc.1202480
  11. Bottger A, Bottger V, Garcia-Echeverria C, Chene P, Hochkeppel HK, Samson W, Ang K, Howard SF, Picksley S.M and Lane DP (1997) Molecular characterization of the hdm2-p53 interaction, J Mol Biol 269: 744-756 https://doi.org/10.1006/jmbi.1997.1078
  12. Canman CE, Wolff AC, Chen CY, Fornance AJ. Jr. and Kastan MB (1994) The p53-dependent G1 cell cycle checkpoint pathway and ataxia-telangiectasia, Cancer Res 54: 5054-5058
  13. Canman CE, Lim DS, Cimprich KA, Taya Y, Tami K, Sakaguchi K, Appella E, Kastan MB and Siliciano JD (1998) Activation of the ATM kinase by ionizing radiation and phosphorylation, Science 281: 1677-1679 https://doi.org/10.1126/science.281.5383.1677
  14. Chaturvedi P, Eng WK, Zhu Y, Mattern MR, Mishra R, Hurle MR, Zhang X, Annan RS, Lu Q, Faucette LF, Scott GF, Li X, Carr SA, Johnson RK, Winkler JD and Zhou BB (1999) Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway, Oncogene 18: 4047-4054 https://doi.org/10.1038/sj.onc.1202925
  15. Chehab NH, Malikzay A, Stavridi ES and Halazoneits TD (1999) Phosphorylation of Ser-20 mediates stabilization of human p53 in response to DNA damage, Proc Natl Acad Sci USA 96: 13777-13782 https://doi.org/10.1073/pnas.96.24.13777
  16. Chehab NH, Malikzay A, Appel M, and Halazonetis TD (2000) Chk2/hCds 1 functions as a DNA damage checkpoint in G(1) by stabilizing p53, Genes Dev 14: 278-288
  17. Dumaz N and Meek DW (1999) Serine 15 phosphorylation stimulate p53 transactivation but does not directly influence interaction with HDM2, EMBO J 18: 7002-7010 https://doi.org/10.1093/emboj/18.24.7002
  18. Falck J, Mailand N, Syljuasen RG, Bartek J and Lukas J (2001) The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis, Nature 410: 842-847 https://doi.org/10.1038/35071124
  19. Farmer G, Bargonetti J, Jhu H, Friedman R, Prywes R and Prives C (1992) Wild-type p53 activities transcription in vitro, Natures 358: 83-86 https://doi.org/10.1038/358083a0
  20. Frische M, Hasseler C and Brandner G (1993) Induction of nuclear accumulation of the tumor-suppressor protein p53 by DNA-damaging agents, Oncogene 8: 307-318
  21. Fu L and Benchimol S (1997) Participation of the human p53 3' UTR translational repression and activation following gamma-irradiation, EMBO J 16: 4117-4125 https://doi.org/10.1093/emboj/16.13.4117
  22. Furnari B, Blasina A, Boddy MN, NcGowan CH, and Russell P (1999) Cdc25 inhibited in vivo and in vitro by checkpoint kinases Cds 1 and Chk1, Mol Cell Biol 10: 833-845 https://doi.org/10.1091/mbc.10.4.833
  23. Giaccia AJ and Kastan MB (1998) The complexity of p53 modulation: emerging patterns from divergent signals, Genes Dev 12: 2973-2983 https://doi.org/10.1101/gad.12.19.2973
  24. Haupt Y, Maya R, Kazaz A and Oren M (1997) Mdm2 promotes the rapid degradation of p53, Nature 387: 296-299 https://doi.org/10.1038/387296a0
  25. Hirao A, Kong YY, Matsuoka S, Wakeham A, Ruland J, Yoshida H, Liu D, Elledge SJ and Mak T (2000) DNA damage-induced activation of p53 by the checkpoint kinase ChK2, Sicence 287: 1824-1827 https://doi.org/10.1126/science.287.5459.1824
  26. Hoess RH and Abremski K (1990) Cre-lox recombination system, Nucleic acids and molecular biology 4: 99-109 https://doi.org/10.1007/978-3-642-84150-7_6
  27. Hollstein M, Shomer B, Greenblatt M, Soussi T, Hovig E, Montesano R and Harris CC (1996) Somatic point mutations in the p53 gene of human tumors and cell lines: updated compilation, Nucleic Acids Res 24: 141-146 https://doi.org/10.1093/nar/24.1.141
  28. Jacks T, Remington L, Williams BO, Schimitt EM, Halachmi S, Bronson RT and Weinberg RA (1994) Tumor spectrum analysis in p53-mutant mice, Curr Biol 4: 1-17 https://doi.org/10.1016/S0960-9822(00)00002-6
  29. Kapoor M and Lozano G (1988) Functional activation of p53 via phosphorylation following DNA damage by UV but not gamma radiation, Proc Natl Acad Sci USA 95: 2834-2837 https://doi.org/10.1073/pnas.95.6.2834
  30. Kastan MB and Lozano G (1998) Participation of p53 protein in the cellular response to DNA damage, Cancer Res 51: 6304-6311
  31. Kastan MB, Zhan Q, El-Deiry WS, Carrier F, Jacks T, Walsh WV, Plukett BS, Vogelstein B and Fornace AJ, Jr. (1992) A mammalian cell cylce checkpoint pathway utilizing p53 amd GADD45 is defective in ataxia-telangiectasia, Cell 71: 587-597 https://doi.org/10.1016/0092-8674(92)90593-2
  32. Kern SE, Pietenpol JA ,Thiagalingam S, Seymour A, Kinzler KW and Vogelstein B (1992) Oncogenic forms of p53 inhibit p53-regulated gene expression, Science 256: 827-830 https://doi.org/10.1126/science.1589764
  33. Khanna KK and Lavin MF (1993) Ionizing radiaton and UV induction of p53 protein by different pathways in ataxiatelangiectasia cells, Oncogene 8: 3307-3312
  34. Khanna KK, Keating KE, Kozolov S, Scot S, Gatei M, Hobson K, Taya Y, Gabrie lli B, Chan D, Lees-Miller SP and Lavin MF (1998) ATM associates with and phosphorylates p53: mapping the region of interaction, Nature Genet 20: 398-400 https://doi.org/10.1038/3882
  35. Knippschild U, Milne DM, Campbell LE, DeMaggio AJ, Christenson E, Hoekstra MF and Meek DW (1997) p53 is phosphorylated in vitro and in vivo by the delta and epsilon isoforms of casein kinase 1 and enhances the level of casein kinase 1 delta in response to topoisomerase-directed drugs, Oncogene 15: 1727-1736 https://doi.org/10.1038/sj.onc.1201541
  36. Ko LJ and Prives C (1996) p53 : puzzle and paradigm, Genes Dev 10: 1054-1072 https://doi.org/10.1101/gad.10.9.1054
  37. Ko LJ, Shieh SY, Chen X, Jayaraman L, Tamai K, Taya Y, Prives C and Pan ZQ (1997) p53 is phosphorylated by CDK7-cyclin H in a p36MAT1-dependent manner, Mol Cell Biol 17: 7220-7229
  38. Kubbutat MH, Jones SN and Vousden KH (1997) Regulation of p53 stability by Mdm2, Nature 387: 290-303 https://doi.org/10.1038/387299a0
  39. Kussie PH, Gorina S, Marechal V, Elenbaas B, Moreau J, Levine AJ and Pavletich NP (1996) Structure of the MDM2 oncoprotein bound to the p53 tumor sppressor transactivation domain, Science 274: 948-953 https://doi.org/10.1126/science.274.5289.948
  40. Lees-Miller SP, Sakaguchi K, Ullrich SJ, Appella E and Anderson CW (1992) Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53, Mol Cell Biol 12: 5041
  41. Levine, AJ (1997) p53, the cellular gatekeeper for growth and division, Cell 88: 323-331 https://doi.org/10.1016/S0092-8674(00)81871-1
  42. Li M, Chen D, Shiloh A, Luo J, Nikolaev AY, Qin J and Gu W (2002) Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization, Nature 416: 648-653 https://doi.org/10.1038/nature737
  43. Liu L, Scolnick DM, Trievel RC, Zhang HB, Marmorstein R, Halazoneits TD, and Berger SL (1999) p53 sites acetylated in vitro by PCAF and p300 are acetylated in vivo in response to DNA damage, Mol Cell Biol 19: 1202-1209
  44. Ljungman, M (2000) Dial 9-1-1 for p53: mechanisms of p53 activation by cellular stress, Neoplasia 2: 208-225 https://doi.org/10.1038/sj.neo.7900073
  45. Lu H, Taya Y, Ikeda M and Levine AJ (1998) Ultraviolet radiation, but not gamma radiation or etoposide-induced DNA damage, results in the phosphorylation of the murine p53 protein at serine-389, Proc Natl Acad Sci USA 95: 6399-6402 https://doi.org/10.1073/pnas.95.11.6399
  46. Maltzman W and Czyzyk L (1984) UV irradiation stimulates levels of p53 cellular tumor antigen in nontransformed mouse cells, Mol Cell Biol 4: 1689-1694
  47. Matsuoka S, Huang M and Elledge SJ (1998) Linkage of ATM to cell cycle regulation by the Chk2 protein kinase, Science 282: 1893-1897 https://doi.org/10.1126/science.282.5395.1893
  48. Matsuoka S, Rotman G, Ogawa A, Shiloh Y, Tamai K, and Elledge SJ (2000) Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro, Proc Natl Acad Sci USA 97: 10389-10394 https://doi.org/10.1073/pnas.190030497
  49. Mayo LD, Turchi JJ, and Berberich SJ (1997) Mdm-2 phosphorylation by DNA-dependent protein kinase prevents interaction with p53, Cancer Res 57: 5013-5016
  50. Meek DW (1999) Mechanisms of switching on p53: a role for covalent modification?, Oncogene 18: 7666-7675 https://doi.org/10.1038/sj.onc.1202951
  51. Melchionna R, Chen XB, Blasina A and McGowan CH (2000) Threonine 68 is required for radiation-induced phosphorylation and activation of Cds1, Nat Cell Biol 2: 762-765 https://doi.org/10.1038/35036406
  52. Midgley CA and Lane DP (1997) p53 protein stability in tumour cells is not determined by mutation but is dependent on Mdm2 binding, Oncogenes 15: 1179-1189 https://doi.org/10.1038/sj.onc.1201459
  53. Milne DM, Palmer RH, Campbell DG, and Meek DW (1992) Phosphorylation of the p53 tumor suppressor protein at three N-termianl sites by a novel casein kinase I-like enzyme, Oncogene 7: 1361-1369
  54. Milne DM, Campbell LE, Campbell DG and Meek DW (1995) p53 is phosphorylated in vitro and in vivo by an ultraviolet radiation-induced protein kinase characteristic of the c-Jun kinase, J Biol Chem 270: 5511-5518 https://doi.org/10.1074/jbc.270.10.5511
  55. Mosner J, Mummenbrauer T, Bauer C, Sczakiel G, Grosse F and Depert W (1995) Negative feedback regulation of wild-type p53 biosynthesis, EMBO J 14: 4442-4449
  56. Oliner JD, Pietenpol JA, Thiagalingam S, Gyuris J, Kinzler KW, and Vogelstein B (1993) Oncoprotein MDM2 conceals the activation domain of tumor suppressor p53, Nature 362: 857-860 https://doi.org/10.1038/362857a0
  57. Oren M,Bienz B,Givol D, Rechavi G and Zakut R (1983) Analysis of recombinant DNA clones specific for the murine p53 cellular tumor antigen, EMBO J 2: 1633-1639
  58. Prives C and Hall PA (1999) The p53 pathway, J Pathol 187: 112-126 https://doi.org/10.1002/(SICI)1096-9896(199901)187:1<112::AID-PATH250>3.0.CO;2-3
  59. Sambrook J, Fritsch EF and Maniatis T (1989) Molecular Cloning: a Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, New Yok
  60. Sakaguchi K, Herrera JE, Saito S, Miko T, Bustin M, Vassilev A, Anderson CW, and Appella E (1998) DNA damage activates p53 through a phosphorylation-acetylation cascade, Genes Dev 12: 2831-2841 https://doi.org/10.1101/gad.12.18.2831
  61. Savitsky K, Bar-Shira A, Gilad S, Rotman G, Ziv Y, Vanagaite L, Tangle DA, Smith S, Uziel T, Sfez S, et al., (1995) A single ataxia telangiectasia gene with a product similar to PI-3 kinase, Science 268: 1749-1753 https://doi.org/10.1126/science.7792600
  62. Shieh SY;Ikeda M;Taya Y;Prives C (1997) DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2, Cell 91: 325-334 https://doi.org/10.1016/S0092-8674(00)80416-X
  63. Shieh SY, Taya Y and Prives C (1999) DNA damage-inducible phosphorylation of p53 at N-terminal sites including a novel site, Ser20, requires tetramerization, EMBO J 18: 1815-1823 https://doi.org/10.1093/emboj/18.7.1815
  64. Shieh SY, Ahn J, Tamai K, Taka Y, and Prives C (2000) The human homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at multiple DNA damage-inducible sites, Genes Dev 14: 289-300
  65. Siliciano JD, Canman CE, Taya Y, Sakaguchi K, Appella E, and Kastan MB (1997) DNA damage induces phosphorylation of the amino terminus of p53, Genes Dev 11: 3471-3481 https://doi.org/10.1101/gad.11.24.3471
  66. Sternberg N (1979) Demonstration and analysis of p1 sitespecific recombination using lambda-P1 hybrid phages constructed in vitro, Quant Biol 43: 1143-1146 https://doi.org/10.1101/SQB.1979.043.01.128
  67. Sternberg N, Sauer B, Hoess R and Abremski K (1986) Bacteriophage P1 cre gene and its regulatory region. Evidence for multiple promoters and for regulation by DNA methylation, J Mol Biol 187: 197-212 https://doi.org/10.1016/0022-2836(86)90228-7
  68. Tibbetts RS, Brumbaugh KM, Williams JM, Sarkaria JN, Cliby WA, Shieh SY, Taya Y, Prives C and Abraham RT (1999) A role for ATR in the DNA damage-induced phosphorylation of p53, Genes Dev 13: 152-157 https://doi.org/10.1101/gad.13.2.152
  69. Tominaga K, Morisaki H, Kaneko Y, Fujimoto A, Takata T, Ohtsubo M and Hirai M (1999) Role of human Cds1 (Chk2) kinase in DNA damage checkpoint and its regulation by p53, J Biol Chem 274: 31463-31467 https://doi.org/10.1074/jbc.274.44.31463
  70. Uesugi M and Verdine GL (1999) The alpha-helical FXXPhiPhi motif in p53: TAF interaction and discrimination by MDM2, Proc Natl Acad Sci USA 96: 14801-14806 https://doi.org/10.1073/pnas.96.26.14801
  71. Unger T, Sionov RV, Moallem E, Yee CL, Howley PM, Oren M and Haupt Y (1999) Mutation in serines 15 and 20 of human p53 impair its apoptic activity, Oncogene 18: 3205-3212 https://doi.org/10.1038/sj.onc.1202656
  72. Waterman MJ, Stavridi ES, Waterman JL and Halazonetis TD (1998) ATM-dependent activation of p53 invovles dephosphorylation and association with 14-3-3 proteins, Nat Genet 19: 175-178 https://doi.org/10.1038/542
  73. Zambetti GP, Bargonneti J, Walker K, Prives C and Levine A (1992) Wild-type p53 mediates positive regulation of gene expression through a specific DNA sequence element, Genes Dev 6: 1143-1152 https://doi.org/10.1101/gad.6.7.1143