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. 1999 Sep;81(2):212–218. doi: 10.1038/sj.bjc.6690679

Proteolytic cleavage of p53 mutants in response to mismatched DNA

T Mee 1, A L Okorokov 1, S Metcalfe 2, J Milner 1
PMCID: PMC2362880  PMID: 10496344

Abstract

Interaction of p53 with mismatched DNA induces proteolytic cleavage with release of a 35-kDa protein fragment from the p53–DNA complexes. The 35-kDa cleavage product is activated for specific biochemical function(s) and may play a role in the cellular response to DNA damage (Molinari et al (1996) Oncogene13: 2077–2086; Okorokov et al (1997) EMBO J16: 6008–6017). In the present study we have asked if mutants of p53 retain the ability to undergo similar proteolytic cleavage, and compared sequence-specific ‘DNA contact’ with ‘structural’ mutants commonly found in human cancer. In addition, a series of phosphorylation site mutants were generated to investigate the possible effects of phosphorylation/dephosphorylation on the proteolytic cleavage of p53. All mutants tested bound to a mismatched DNA target in vitro. Moreover, studies in vitro and in vivo indicate that p53 mutants with intact conformational structure (as determined by immunoreactivity with PAb246 and PAb1620) retain the ability to undergo proteolytic cleavage similar, if not identical, to the wild-type p53 protein. Our results suggest that the capacity for p53 to bind mismatched DNA is independent of structural conformation of the central core domain. Proteolytic cleavage, however, is crucially dependent upon a wild-type conformation of the protein. © 1999 Cancer Research Campaign

Keywords: p53 mutants, proteolytic cleavage, mismatched DNA

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Selected References

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  1. Caelles C., Helmberg A., Karin M. p53-dependent apoptosis in the absence of transcriptional activation of p53-target genes. Nature. 1994 Jul 21;370(6486):220–223. doi: 10.1038/370220a0. [DOI] [PubMed] [Google Scholar]
  2. Cho Y., Gorina S., Jeffrey P. D., Pavletich N. P. Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science. 1994 Jul 15;265(5170):346–355. doi: 10.1126/science.8023157. [DOI] [PubMed] [Google Scholar]
  3. Funk W. D., Pak D. T., Karas R. H., Wright W. E., Shay J. W. A transcriptionally active DNA-binding site for human p53 protein complexes. Mol Cell Biol. 1992 Jun;12(6):2866–2871. doi: 10.1128/mcb.12.6.2866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gannon J. V., Greaves R., Iggo R., Lane D. P. Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant form. EMBO J. 1990 May;9(5):1595–1602. doi: 10.1002/j.1460-2075.1990.tb08279.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gorina S., Pavletich N. P. Structure of the p53 tumor suppressor bound to the ankyrin and SH3 domains of 53BP2. Science. 1996 Nov 8;274(5289):1001–1005. doi: 10.1126/science.274.5289.1001. [DOI] [PubMed] [Google Scholar]
  6. Gottlieb T. M., Oren M. p53 in growth control and neoplasia. Biochim Biophys Acta. 1996 Jun 7;1287(2-3):77–102. doi: 10.1016/0304-419x(95)00019-c. [DOI] [PubMed] [Google Scholar]
  7. Haupt Y., Oren M. p53-mediated apoptosis: mechanisms and regulation. Behring Inst Mitt. 1996 Oct;(97):32–59. [PubMed] [Google Scholar]
  8. Haupt Y., Rowan S., Shaulian E., Vousden K. H., Oren M. Induction of apoptosis in HeLa cells by trans-activation-deficient p53. Genes Dev. 1995 Sep 1;9(17):2170–2183. doi: 10.1101/gad.9.17.2170. [DOI] [PubMed] [Google Scholar]
  9. Jayaraman J., Prives C. Activation of p53 sequence-specific DNA binding by short single strands of DNA requires the p53 C-terminus. Cell. 1995 Jun 30;81(7):1021–1029. doi: 10.1016/s0092-8674(05)80007-8. [DOI] [PubMed] [Google Scholar]
  10. Ko L. J., Prives C. p53: puzzle and paradigm. Genes Dev. 1996 May 1;10(9):1054–1072. doi: 10.1101/gad.10.9.1054. [DOI] [PubMed] [Google Scholar]
  11. Lee S., Elenbaas B., Levine A., Griffith J. p53 and its 14 kDa C-terminal domain recognize primary DNA damage in the form of insertion/deletion mismatches. Cell. 1995 Jun 30;81(7):1013–1020. doi: 10.1016/s0092-8674(05)80006-6. [DOI] [PubMed] [Google Scholar]
  12. Levine A. J. p53, the cellular gatekeeper for growth and division. Cell. 1997 Feb 7;88(3):323–331. doi: 10.1016/s0092-8674(00)81871-1. [DOI] [PubMed] [Google Scholar]
  13. Meek D. W. Post-translational modification of p53. Semin Cancer Biol. 1994 Jun;5(3):203–210. [PubMed] [Google Scholar]
  14. Michalovitz D., Halevy O., Oren M. Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant of p53. Cell. 1990 Aug 24;62(4):671–680. doi: 10.1016/0092-8674(90)90113-s. [DOI] [PubMed] [Google Scholar]
  15. Milne D. M., McKendrick L., Jardine L. J., Deacon E., Lord J. M., Meek D. W. Murine p53 is phosphorylated within the PAb421 epitope by protein kinase C in vitro, but not in vivo, even after stimulation with the phorbol ester o-tetradecanoylphorbol 13-acetate. Oncogene. 1996 Jul 4;13(1):205–211. [PubMed] [Google Scholar]
  16. Milner J., Medcalf E. A., Cook A. C. Tumor suppressor p53: analysis of wild-type and mutant p53 complexes. Mol Cell Biol. 1991 Jan;11(1):12–19. doi: 10.1128/mcb.11.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Miyashita T., Reed J. C. Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell. 1995 Jan 27;80(2):293–299. doi: 10.1016/0092-8674(95)90412-3. [DOI] [PubMed] [Google Scholar]
  18. Molinari M., Okorokov A. L., Milner J. Interaction with damaged DNA induces selective proteolytic cleavage of p53 to yield 40 kDa and 35 kDa fragments competent for sequence-specific DNA binding. Oncogene. 1996 Nov 21;13(10):2077–2086. [PubMed] [Google Scholar]
  19. Mummenbrauer T., Janus F., Müller B., Wiesmüller L., Deppert W., Grosse F. p53 Protein exhibits 3'-to-5' exonuclease activity. Cell. 1996 Jun 28;85(7):1089–1099. doi: 10.1016/s0092-8674(00)81309-4. [DOI] [PubMed] [Google Scholar]
  20. Okorokov A. L., Ponchel F., Milner J. Induced N- and C-terminal cleavage of p53: a core fragment of p53, generated by interaction with damaged DNA, promotes cleavage of the N-terminus of full-length p53, whereas ssDNA induces C-terminal cleavage of p53. EMBO J. 1997 Oct 1;16(19):6008–6017. doi: 10.1093/emboj/16.19.6008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Paetzel M., Dalbey R. E. Catalytic hydroxyl/amine dyads within serine proteases. Trends Biochem Sci. 1997 Jan;22(1):28–31. doi: 10.1016/s0968-0004(96)10065-7. [DOI] [PubMed] [Google Scholar]
  22. Steegenga W. T., van der Eb A. J., Jochemsen A. G. How phosphorylation regulates the activity of p53. J Mol Biol. 1996 Oct 25;263(2):103–113. doi: 10.1006/jmbi.1996.0560. [DOI] [PubMed] [Google Scholar]
  23. Takenaka I., Morin F., Seizinger B. R., Kley N. Regulation of the sequence-specific DNA binding function of p53 by protein kinase C and protein phosphatases. J Biol Chem. 1995 Mar 10;270(10):5405–5411. doi: 10.1074/jbc.270.10.5405. [DOI] [PubMed] [Google Scholar]
  24. Thukral S. K., Blain G. C., Chang K. K., Fields S. Distinct residues of human p53 implicated in binding to DNA, simian virus 40 large T antigen, 53BP1, and 53BP2. Mol Cell Biol. 1994 Dec;14(12):8315–8321. doi: 10.1128/mcb.14.12.8315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. el-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. WAF1, a potential mediator of p53 tumor suppression. Cell. 1993 Nov 19;75(4):817–825. doi: 10.1016/0092-8674(93)90500-p. [DOI] [PubMed] [Google Scholar]

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