Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1994 Oct 17;13(20):4823–4830. doi: 10.1002/j.1460-2075.1994.tb06808.x

Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells.

Y Wu 1, Y Liu 1, L Lee 1, Z Miner 1, M Kulesz-Martin 1
PMCID: PMC395421  PMID: 7957051

Abstract

A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence-specific DNA-binding ability. DNA binding by p53 protein is inefficient in comparison and requires activation. Furthermore, p53as and p53 proteins formed hetero-oligomers when co-translated in vitro, resulting in inactivation of p53as DNA-binding activity. Gel filtration indicated that p53as translated in vitro, like p53, formed tetramers. In support of a functional role of p53as in cells, p53as/p53 hetero-oligomers were coimmunoprecipitated from mouse cells, and both protein forms were detectable in nuclear extracts by electrophoretic mobility shift assays. These results suggest that the biochemical functions of p53 are mediated by interaction between two endogenous protein products of the wild-type p53 gene.

Full text

PDF
4823

Images in this article

4824Image
on p.4824
4825Image
on p.4825
4826Image
on p.4826
4827Image
on p.4827
4827Image
on p.4827
4828Image
on p.4828

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Arai N., Nomura D., Yokota K., Wolf D., Brill E., Shohat O., Rotter V. Immunologically distinct p53 molecules generated by alternative splicing. Mol Cell Biol. 1986 Sep;6(9):3232–3239. doi: 10.1128/mcb.6.9.3232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barak Y., Juven T., Haffner R., Oren M. mdm2 expression is induced by wild type p53 activity. EMBO J. 1993 Feb;12(2):461–468. doi: 10.1002/j.1460-2075.1993.tb05678.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bargonetti J., Friedman P. N., Kern S. E., Vogelstein B., Prives C. Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication. Cell. 1991 Jun 14;65(6):1083–1091. doi: 10.1016/0092-8674(91)90560-l. [DOI] [PubMed] [Google Scholar]
  4. Bargonetti J., Manfredi J. J., Chen X., Marshak D. R., Prives C. A proteolytic fragment from the central region of p53 has marked sequence-specific DNA-binding activity when generated from wild-type but not from oncogenic mutant p53 protein. Genes Dev. 1993 Dec;7(12B):2565–2574. doi: 10.1101/gad.7.12b.2565. [DOI] [PubMed] [Google Scholar]
  5. Farmer G., Bargonetti J., Zhu H., Friedman P., Prywes R., Prives C. Wild-type p53 activates transcription in vitro. Nature. 1992 Jul 2;358(6381):83–86. doi: 10.1038/358083a0. [DOI] [PubMed] [Google Scholar]
  6. Finlay C. A. The mdm-2 oncogene can overcome wild-type p53 suppression of transformed cell growth. Mol Cell Biol. 1993 Jan;13(1):301–306. doi: 10.1128/mcb.13.1.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Friedman P. N., Chen X., Bargonetti J., Prives C. The p53 protein is an unusually shaped tetramer that binds directly to DNA. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3319–3323. doi: 10.1073/pnas.90.8.3319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Gu Y., Turck C. W., Morgan D. O. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature. 1993 Dec 16;366(6456):707–710. doi: 10.1038/366707a0. [DOI] [PubMed] [Google Scholar]
  10. Hainaut P., Milner J. Interaction of heat-shock protein 70 with p53 translated in vitro: evidence for interaction with dimeric p53 and for a role in the regulation of p53 conformation. EMBO J. 1992 Oct;11(10):3513–3520. doi: 10.1002/j.1460-2075.1992.tb05434.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Halazonetis T. D., Davis L. J., Kandil A. N. Wild-type p53 adopts a 'mutant'-like conformation when bound to DNA. EMBO J. 1993 Mar;12(3):1021–1028. doi: 10.1002/j.1460-2075.1993.tb05743.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Han K. A., Kulesz-Martin M. F. Alternatively spliced p53 RNA in transformed and normal cells of different tissue types. Nucleic Acids Res. 1992 Apr 25;20(8):1979–1981. doi: 10.1093/nar/20.8.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
  14. Hupp T. R., Meek D. W., Midgley C. A., Lane D. P. Regulation of the specific DNA binding function of p53. Cell. 1992 Nov 27;71(5):875–886. doi: 10.1016/0092-8674(92)90562-q. [DOI] [PubMed] [Google Scholar]
  15. Juven T., Barak Y., Zauberman A., George D. L., Oren M. Wild type p53 can mediate sequence-specific transactivation of an internal promoter within the mdm2 gene. Oncogene. 1993 Dec;8(12):3411–3416. [PubMed] [Google Scholar]
  16. Kastan M. B., Onyekwere O., Sidransky D., Vogelstein B., Craig R. W. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991 Dec 1;51(23 Pt 1):6304–6311. [PubMed] [Google Scholar]
  17. Kastan M. B., Zhan Q., el-Deiry W. S., Carrier F., Jacks T., Walsh W. V., Plunkett B. S., Vogelstein B., Fornace A. J., Jr A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in ataxia-telangiectasia. Cell. 1992 Nov 13;71(4):587–597. doi: 10.1016/0092-8674(92)90593-2. [DOI] [PubMed] [Google Scholar]
  18. Kern S. E., Kinzler K. W., Bruskin A., Jarosz D., Friedman P., Prives C., Vogelstein B. Identification of p53 as a sequence-specific DNA-binding protein. Science. 1991 Jun 21;252(5013):1708–1711. doi: 10.1126/science.2047879. [DOI] [PubMed] [Google Scholar]
  19. Kern S. E., Pietenpol J. A., Thiagalingam S., Seymour A., Kinzler K. W., Vogelstein B. Oncogenic forms of p53 inhibit p53-regulated gene expression. Science. 1992 May 8;256(5058):827–830. doi: 10.1126/science.1589764. [DOI] [PubMed] [Google Scholar]
  20. Kuerbitz S. J., Plunkett B. S., Walsh W. V., Kastan M. B. Wild-type p53 is a cell cycle checkpoint determinant following irradiation. Proc Natl Acad Sci U S A. 1992 Aug 15;89(16):7491–7495. doi: 10.1073/pnas.89.16.7491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kulesz-Martin M. F., Lisafeld B., Huang H., Kisiel N. D., Lee L. Endogenous p53 protein generated from wild-type alternatively spliced p53 RNA in mouse epidermal cells. Mol Cell Biol. 1994 Mar;14(3):1698–1708. doi: 10.1128/mcb.14.3.1698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lane D. P. Cancer. p53, guardian of the genome. Nature. 1992 Jul 2;358(6381):15–16. doi: 10.1038/358015a0. [DOI] [PubMed] [Google Scholar]
  23. Lassar A. B., Davis R. L., Wright W. E., Kadesch T., Murre C., Voronova A., Baltimore D., Weintraub H. Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo. Cell. 1991 Jul 26;66(2):305–315. doi: 10.1016/0092-8674(91)90620-e. [DOI] [PubMed] [Google Scholar]
  24. Milner J., Medcalf E. A. Cotranslation of activated mutant p53 with wild type drives the wild-type p53 protein into the mutant conformation. Cell. 1991 May 31;65(5):765–774. doi: 10.1016/0092-8674(91)90384-b. [DOI] [PubMed] [Google Scholar]
  25. Momand J., Zambetti G. P., Olson D. C., George D., Levine A. J. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell. 1992 Jun 26;69(7):1237–1245. doi: 10.1016/0092-8674(92)90644-r. [DOI] [PubMed] [Google Scholar]
  26. Oliner J. D., Kinzler K. W., Meltzer P. S., George D. L., Vogelstein B. Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature. 1992 Jul 2;358(6381):80–83. doi: 10.1038/358080a0. [DOI] [PubMed] [Google Scholar]
  27. Pavletich N. P., Chambers K. A., Pabo C. O. The DNA-binding domain of p53 contains the four conserved regions and the major mutation hot spots. Genes Dev. 1993 Dec;7(12B):2556–2564. doi: 10.1101/gad.7.12b.2556. [DOI] [PubMed] [Google Scholar]
  28. Schärer E., Iggo R. Mammalian p53 can function as a transcription factor in yeast. Nucleic Acids Res. 1992 Apr 11;20(7):1539–1545. doi: 10.1093/nar/20.7.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Serrano M., Hannon G. J., Beach D. A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4. Nature. 1993 Dec 16;366(6456):704–707. doi: 10.1038/366704a0. [DOI] [PubMed] [Google Scholar]
  30. Shaulian E., Zauberman A., Milner J., Davies E. A., Oren M. Tight DNA binding and oligomerization are dispensable for the ability of p53 to transactivate target genes and suppress transformation. EMBO J. 1993 Jul;12(7):2789–2797. doi: 10.1002/j.1460-2075.1993.tb05940.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Stürzbecher H. W., Brain R., Addison C., Rudge K., Remm M., Grimaldi M., Keenan E., Jenkins J. R. A C-terminal alpha-helix plus basic region motif is the major structural determinant of p53 tetramerization. Oncogene. 1992 Aug;7(8):1513–1523. [PubMed] [Google Scholar]
  32. Wang Y., Reed M., Wang P., Stenger J. E., Mayr G., Anderson M. E., Schwedes J. F., Tegtmeyer P. p53 domains: identification and characterization of two autonomous DNA-binding regions. Genes Dev. 1993 Dec;7(12B):2575–2586. doi: 10.1101/gad.7.12b.2575. [DOI] [PubMed] [Google Scholar]
  33. Wolf D., Harris N., Goldfinger N., Rotter V. Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule. Mol Cell Biol. 1985 Jan;5(1):127–132. doi: 10.1128/mcb.5.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Xiong Y., Hannon G. J., Zhang H., Casso D., Kobayashi R., Beach D. p21 is a universal inhibitor of cyclin kinases. Nature. 1993 Dec 16;366(6456):701–704. doi: 10.1038/366701a0. [DOI] [PubMed] [Google Scholar]
  35. Zambetti G. P., Bargonetti J., Walker K., Prives C., Levine A. J. Wild-type p53 mediates positive regulation of gene expression through a specific DNA sequence element. Genes Dev. 1992 Jul;6(7):1143–1152. doi: 10.1101/gad.6.7.1143. [DOI] [PubMed] [Google Scholar]
  36. Zauberman A., Barak Y., Ragimov N., Levy N., Oren M. Sequence-specific DNA binding by p53: identification of target sites and lack of binding to p53 - MDM2 complexes. EMBO J. 1993 Jul;12(7):2799–2808. doi: 10.1002/j.1460-2075.1993.tb05941.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. el-Deiry W. S., Kern S. E., Pietenpol J. A., Kinzler K. W., Vogelstein B. Definition of a consensus binding site for p53. Nat Genet. 1992 Apr;1(1):45–49. doi: 10.1038/ng0492-45. [DOI] [PubMed] [Google Scholar]
  38. 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]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES