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. 1992 Jun;11(6):2115–2121. doi: 10.1002/j.1460-2075.1992.tb05270.x

Enhanced binding of a 95 kDa protein to p53 in cells undergoing p53-mediated growth arrest.

Y Barak 1, M Oren 1
PMCID: PMC556678  PMID: 1600943

Abstract

To explore the biochemical functions of p53, we have initiated a search for cellular p53-binding proteins. Coprecipitation of three polypeptides was observed when cell lines overexpressing a temperature-sensitive (ts) p53 mutant were maintained at 32.5 degrees C (wild-type p53 activity, leading to growth arrest) but not at 37.5 degrees C (mutant p53 activity). One of these three proteins, designated p95 on the basis of its apparent molecular mass, was highly abundant in p53 immune complexes. We demonstrate herein that p95 is a p53-binding protein, which exhibits poor p53-binding in cells overproducing several distinct mutant p53 proteins. Yet, p95 associates equally well with both the wild-type (wt) and the mutant conformations of the ts p53 in transformed cells growth-arrested at 32.5 degrees C. On the basis of our findings we suggest that wt p53 activity increases p53-p95 complex formation and that such interaction may play a central role in p53 mediated tumour suppression.

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Selected References

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

  1. Bagchi S., Weinmann R., Raychaudhuri P. The retinoblastoma protein copurifies with E2F-I, an E1A-regulated inhibitor of the transcription factor E2F. Cell. 1991 Jun 14;65(6):1063–1072. doi: 10.1016/0092-8674(91)90558-g. [DOI] [PubMed] [Google Scholar]
  2. Baker S. J., Markowitz S., Fearon E. R., Willson J. K., Vogelstein B. Suppression of human colorectal carcinoma cell growth by wild-type p53. Science. 1990 Aug 24;249(4971):912–915. doi: 10.1126/science.2144057. [DOI] [PubMed] [Google Scholar]
  3. Bandara L. R., La Thangue N. B. Adenovirus E1a prevents the retinoblastoma gene product from complexing with a cellular transcription factor. Nature. 1991 Jun 6;351(6326):494–497. doi: 10.1038/351494a0. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Braithwaite A. W., Sturzbecher H. W., Addison C., Palmer C., Rudge K., Jenkins J. R. Mouse p53 inhibits SV40 origin-dependent DNA replication. Nature. 1987 Oct 1;329(6138):458–460. doi: 10.1038/329458a0. [DOI] [PubMed] [Google Scholar]
  6. Chellappan S. P., Hiebert S., Mudryj M., Horowitz J. M., Nevins J. R. The E2F transcription factor is a cellular target for the RB protein. Cell. 1991 Jun 14;65(6):1053–1061. doi: 10.1016/0092-8674(91)90557-f. [DOI] [PubMed] [Google Scholar]
  7. Chittenden T., Livingston D. M., Kaelin W. G., Jr The T/E1A-binding domain of the retinoblastoma product can interact selectively with a sequence-specific DNA-binding protein. Cell. 1991 Jun 14;65(6):1073–1082. doi: 10.1016/0092-8674(91)90559-h. [DOI] [PubMed] [Google Scholar]
  8. Clarke C. F., Cheng K., Frey A. B., Stein R., Hinds P. W., Levine A. J. Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP. Mol Cell Biol. 1988 Mar;8(3):1206–1215. doi: 10.1128/mcb.8.3.1206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Diller L., Kassel J., Nelson C. E., Gryka M. A., Litwak G., Gebhardt M., Bressac B., Ozturk M., Baker S. J., Vogelstein B. p53 functions as a cell cycle control protein in osteosarcomas. Mol Cell Biol. 1990 Nov;10(11):5772–5781. doi: 10.1128/mcb.10.11.5772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Eizenberg O., Oren M. Reduced levels of alpha 1 (I) collagen mRNA in cells immortalized by mutant p53 or transformed by ras. Biochim Biophys Acta. 1991 Dec 2;1129(1):34–42. doi: 10.1016/0167-4781(91)90209-5. [DOI] [PubMed] [Google Scholar]
  11. Eliyahu D., Michalovitz D., Eliyahu S., Pinhasi-Kimhi O., Oren M. Wild-type p53 can inhibit oncogene-mediated focus formation. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8763–8767. doi: 10.1073/pnas.86.22.8763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eliyahu D., Michalovitz D., Oren M. Overproduction of p53 antigen makes established cells highly tumorigenic. Nature. 1985 Jul 11;316(6024):158–160. doi: 10.1038/316158a0. [DOI] [PubMed] [Google Scholar]
  13. Eliyahu D., Raz A., Gruss P., Givol D., Oren M. Participation of p53 cellular tumour antigen in transformation of normal embryonic cells. Nature. 1984 Dec 13;312(5995):646–649. doi: 10.1038/312646a0. [DOI] [PubMed] [Google Scholar]
  14. Fields S., Jang S. K. Presence of a potent transcription activating sequence in the p53 protein. Science. 1990 Aug 31;249(4972):1046–1049. doi: 10.1126/science.2144363. [DOI] [PubMed] [Google Scholar]
  15. Finlay C. A., Hinds P. W., Tan T. H., Eliyahu D., Oren M., Levine A. J. Activating mutations for transformation by p53 produce a gene product that forms an hsc70-p53 complex with an altered half-life. Mol Cell Biol. 1988 Feb;8(2):531–539. doi: 10.1128/mcb.8.2.531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Friedman P. N., Kern S. E., Vogelstein B., Prives C. Wild-type, but not mutant, human p53 proteins inhibit the replication activities of simian virus 40 large tumor antigen. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9275–9279. doi: 10.1073/pnas.87.23.9275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Gannon J. V., Lane D. P. Protein synthesis required to anchor a mutant p53 protein which is temperature-sensitive for nuclear transport. Nature. 1991 Feb 28;349(6312):802–806. doi: 10.1038/349802a0. [DOI] [PubMed] [Google Scholar]
  19. Gannon J. V., Lane D. P. p53 and DNA polymerase alpha compete for binding to SV40 T antigen. Nature. 1987 Oct 1;329(6138):456–458. doi: 10.1038/329456a0. [DOI] [PubMed] [Google Scholar]
  20. Ginsberg D., Mechta F., Yaniv M., Oren M. Wild-type p53 can down-modulate the activity of various promoters. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9979–9983. doi: 10.1073/pnas.88.22.9979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ginsberg D., Michael-Michalovitz D., Ginsberg D., Oren M. Induction of growth arrest by a temperature-sensitive p53 mutant is correlated with increased nuclear localization and decreased stability of the protein. Mol Cell Biol. 1991 Jan;11(1):582–585. doi: 10.1128/mcb.11.1.582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Halevy O., Hall A., Oren M. Stabilization of the p53 transformation-related protein in mouse fibrosarcoma cell lines: effects of protein sequence and intracellular environment. Mol Cell Biol. 1989 Aug;9(8):3385–3392. doi: 10.1128/mcb.9.8.3385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Halevy O., Michalovitz D., Oren M. Different tumor-derived p53 mutants exhibit distinct biological activities. Science. 1990 Oct 5;250(4977):113–116. doi: 10.1126/science.2218501. [DOI] [PubMed] [Google Scholar]
  24. Halevy O., Rodel J., Peled A., Oren M. Frequent p53 mutations in chemically induced murine fibrosarcoma. Oncogene. 1991 Sep;6(9):1593–1600. [PubMed] [Google Scholar]
  25. Hinds P. W., Finlay C. A., Frey A. B., Levine A. J. Immunological evidence for the association of p53 with a heat shock protein, hsc70, in p53-plus-ras-transformed cell lines. Mol Cell Biol. 1987 Aug;7(8):2863–2869. doi: 10.1128/mcb.7.8.2863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hinds P. W., Finlay C. A., Quartin R. S., Baker S. J., Fearon E. R., Vogelstein B., Levine A. J. Mutant p53 DNA clones from human colon carcinomas cooperate with ras in transforming primary rat cells: a comparison of the "hot spot" mutant phenotypes. Cell Growth Differ. 1990 Dec;1(12):571–580. [PubMed] [Google Scholar]
  27. Hollstein M., Sidransky D., Vogelstein B., Harris C. C. p53 mutations in human cancers. Science. 1991 Jul 5;253(5015):49–53. doi: 10.1126/science.1905840. [DOI] [PubMed] [Google Scholar]
  28. Huang S., Lee W. H., Lee E. Y. A cellular protein that competes with SV40 T antigen for binding to the retinoblastoma gene product. Nature. 1991 Mar 14;350(6314):160–162. doi: 10.1038/350160a0. [DOI] [PubMed] [Google Scholar]
  29. Kaelin W. G., Jr, Pallas D. C., DeCaprio J. A., Kaye F. J., Livingston D. M. Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product. Cell. 1991 Feb 8;64(3):521–532. doi: 10.1016/0092-8674(91)90236-r. [DOI] [PubMed] [Google Scholar]
  30. Kern S. E., Kinzler K. W., Baker S. J., Nigro J. M., Rotter V., Levine A. J., Friedman P., Prives C., Vogelstein B. Mutant p53 proteins bind DNA abnormally in vitro. Oncogene. 1991 Jan;6(1):131–136. [PubMed] [Google Scholar]
  31. 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]
  32. Levine A. J., Momand J., Finlay C. A. The p53 tumour suppressor gene. Nature. 1991 Jun 6;351(6326):453–456. doi: 10.1038/351453a0. [DOI] [PubMed] [Google Scholar]
  33. Levine A. J. The p53 protein and its interactions with the oncogene products of the small DNA tumor viruses. Virology. 1990 Aug;177(2):419–426. doi: 10.1016/0042-6822(90)90505-l. [DOI] [PubMed] [Google Scholar]
  34. Malkin D., Li F. P., Strong L. C., Fraumeni J. F., Jr, Nelson C. E., Kim D. H., Kassel J., Gryka M. A., Bischoff F. Z., Tainsky M. A. Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and other neoplasms. Science. 1990 Nov 30;250(4985):1233–1238. doi: 10.1126/science.1978757. [DOI] [PubMed] [Google Scholar]
  35. Maltzman W., Oren M., Levine A. J. The structural relationships between 54,000-molecular-weight cellular tumor antigens detected in viral- and nonviral-transformed cells. Virology. 1981 Jul 15;112(1):145–156. doi: 10.1016/0042-6822(81)90620-6. [DOI] [PubMed] [Google Scholar]
  36. Martinez J., Georgoff I., Martinez J., Levine A. J. Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein. Genes Dev. 1991 Feb;5(2):151–159. doi: 10.1101/gad.5.2.151. [DOI] [PubMed] [Google Scholar]
  37. Mercer W. E., Shields M. T., Amin M., Sauve G. J., Appella E., Romano J. W., Ullrich S. J. Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6166–6170. doi: 10.1073/pnas.87.16.6166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mercer W. E., Shields M. T., Lin D., Appella E., Ullrich S. J. Growth suppression induced by wild-type p53 protein is accompanied by selective down-regulation of proliferating-cell nuclear antigen expression. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1958–1962. doi: 10.1073/pnas.88.5.1958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. Michalovitz D., Halevy O., Oren M. p53 mutations: gains or losses? J Cell Biochem. 1991 Jan;45(1):22–29. doi: 10.1002/jcb.240450108. [DOI] [PubMed] [Google Scholar]
  41. O'Rourke R. W., Miller C. W., Kato G. J., Simon K. J., Chen D. L., Dang C. V., Koeffler H. P. A potential transcriptional activation element in the p53 protein. Oncogene. 1990 Dec;5(12):1829–1832. [PubMed] [Google Scholar]
  42. Oren M., Levine A. J. Immunoselection of simian virus 40 large T antigen messenger rnas from transformed cells. Virology. 1981 Sep;113(2):790–793. doi: 10.1016/0042-6822(81)90210-5. [DOI] [PubMed] [Google Scholar]
  43. Pinhasi-Kimhi O., Michalovitz D., Ben-Zeev A., Oren M. Specific interaction between the p53 cellular tumour antigen and major heat shock proteins. Nature. 1986 Mar 13;320(6058):182–184. doi: 10.1038/320182a0. [DOI] [PubMed] [Google Scholar]
  44. Raycroft L., Schmidt J. R., Yoas K., Hao M. M., Lozano G. Analysis of p53 mutants for transcriptional activity. Mol Cell Biol. 1991 Dec;11(12):6067–6074. doi: 10.1128/mcb.11.12.6067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Raycroft L., Wu H. Y., Lozano G. Transcriptional activation by wild-type but not transforming mutants of the p53 anti-oncogene. Science. 1990 Aug 31;249(4972):1049–1051. doi: 10.1126/science.2144364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Reihsaus E., Kohler M., Kraiss S., Oren M., Montenarh M. Regulation of the level of the oncoprotein p53 in non-transformed and transformed cells. Oncogene. 1990 Jan;5(1):137–145. [PubMed] [Google Scholar]
  47. Rustgi A. K., Dyson N., Bernards R. Amino-terminal domains of c-myc and N-myc proteins mediate binding to the retinoblastoma gene product. Nature. 1991 Aug 8;352(6335):541–544. doi: 10.1038/352541a0. [DOI] [PubMed] [Google Scholar]
  48. Santhanam U., Ray A., Sehgal P. B. Repression of the interleukin 6 gene promoter by p53 and the retinoblastoma susceptibility gene product. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7605–7609. doi: 10.1073/pnas.88.17.7605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Shaulsky G., Goldfinger N., Peled A., Rotter V. Involvement of wild-type p53 in pre-B-cell differentiation in vitro. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8982–8986. doi: 10.1073/pnas.88.20.8982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Srivastava S., Zou Z. Q., Pirollo K., Blattner W., Chang E. H. Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome. Nature. 1990 Dec 20;348(6303):747–749. doi: 10.1038/348747a0. [DOI] [PubMed] [Google Scholar]
  51. Steinmeyer K., Deppert W. DNA binding properties of murine p53. Oncogene. 1988 Nov;3(5):501–507. [PubMed] [Google Scholar]
  52. Stürzbecher H. W., Addison C., Jenkins J. R. Characterization of mutant p53-hsp72/73 protein-protein complexes by transient expression in monkey COS cells. Mol Cell Biol. 1988 Sep;8(9):3740–3747. doi: 10.1128/mcb.8.9.3740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stürzbecher H. W., Chumakov P., Welch W. J., Jenkins J. R. Mutant p53 proteins bind hsp 72/73 cellular heat shock-related proteins in SV40-transformed monkey cells. Oncogene. 1987 May;1(2):201–211. [PubMed] [Google Scholar]
  54. Weinberg R. A. Tumor suppressor genes. Science. 1991 Nov 22;254(5035):1138–1146. doi: 10.1126/science.1659741. [DOI] [PubMed] [Google Scholar]
  55. Weintraub H., Hauschka S., Tapscott S. J. The MCK enhancer contains a p53 responsive element. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4570–4571. doi: 10.1073/pnas.88.11.4570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wilcock D., Lane D. P. Localization of p53, retinoblastoma and host replication proteins at sites of viral replication in herpes-infected cells. Nature. 1991 Jan 31;349(6308):429–431. doi: 10.1038/349429a0. [DOI] [PubMed] [Google Scholar]
  57. Yewdell J. W., Gannon J. V., Lane D. P. Monoclonal antibody analysis of p53 expression in normal and transformed cells. J Virol. 1986 Aug;59(2):444–452. doi: 10.1128/jvi.59.2.444-452.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yonish-Rouach E., Resnitzky D., Lotem J., Sachs L., Kimchi A., Oren M. Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature. 1991 Jul 25;352(6333):345–347. doi: 10.1038/352345a0. [DOI] [PubMed] [Google Scholar]

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