Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1992 Jun;66(6):3846–3859. doi: 10.1128/jvi.66.6.3846-3859.1992

Species-specific phosphorylation of mouse and rat p53 in simian virus 40-transformed cells.

T Patschinsky 1, U Knippschild 1, W Deppert 1
PMCID: PMC241170  PMID: 1316485

Abstract

We have analyzed in detail the phosphorylation of p53 from normal (3T3) and simian virus 40 (SV40)-transformed (SV3T3) BALB/c mouse cells and from normal (F111) and SV40-transformed [FR(wt648)] rat cells by two-dimensional tryptic peptide mapping and phosphoamino acid analyses. To accommodate the different half-lives of p53 in normal (half-life, 15 min) and transformed (half-life, 20 h) cells and possible differences in the rates of turnover of phosphate at specific sites, cells were labeled for 2 h (short-term labeling) or 18 h (long-term labeling). Depending on the labeling conditions, either close similarities or marked differences were observed in the phosphorylation patterns of p53 from normal and transformed cells. After the 2-h labeling, the phosphorylation patterns of p53 from normal and transformed mouse cells were quite similar. In contrast, p53 from normal and transformed rat cells exhibited dramatic quantitative and qualitative differences under these labeling conditions. The reverse was found after an 18-h label leading to steady-state phosphorylation of p53 in transformed cells: while p53 in transformed mouse cells revealed a marked quantitative increase in phosphorylation compared with p53 from normal cells, the corresponding patterns of p53 from normal and transformed rat cells were similar. Our data thus indicate species-specific differences in the phosphorylation of mouse and rat p53 in SV40-transformed cells, reflected by (i) different turnover rates at specific sites in mouse and rat p53 and (ii) phosphorylation of nonhomologous serine and threonine residues in rat p53, as revealed by indirect assignment of phosphorylation sites to the phosphopeptides of rat p53. Analyses of p53 from the SV40 tsA58 mutant-transformed F111 cell lines FR(tsA58)A (N type) and FR(tsA58)57 (A type) yielded no conclusive evidence for a direct correlation between phosphorylation of p53, the metabolic stabilization of p53, and expression of the transformed phenotype.

Full text

PDF
3846

Images in this article

3849Image
on p.3849
3850Image
on p.3850
3851Image
on p.3851
3852Image
on p.3852
3855Image
on p.3855

Selected References

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

  1. Aaronson S. A., Todaro G. J. Development of 3T3-like lines from Balb-c mouse embryo cultures: transformation susceptibility to SV40. J Cell Physiol. 1968 Oct;72(2):141–148. doi: 10.1002/jcp.1040720208. [DOI] [PubMed] [Google Scholar]
  2. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bischoff J. R., Friedman P. N., Marshak D. R., Prives C., Beach D. Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4766–4770. doi: 10.1073/pnas.87.12.4766. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  5. Cyert M. S., Thorner J. Putting it on and taking it off: phosphoprotein phosphatase involvement in cell cycle regulation. Cell. 1989 Jun 16;57(6):891–893. doi: 10.1016/0092-8674(89)90325-5. [DOI] [PubMed] [Google Scholar]
  6. Deppert W., Buschhausen-Denker G., Patschinsky T., Steinmeyer K. Cell cycle control of p53 in normal (3T3) and chemically transformed (Meth A) mouse cells. II. Requirement for cell cycle progression. Oncogene. 1990 Nov;5(11):1701–1706. [PubMed] [Google Scholar]
  7. Deppert W., Haug M. Evidence for free and metabolically stable p53 protein in nuclear subfractions of simian virus 40-transformed cells. Mol Cell Biol. 1986 Jun;6(6):2233–2240. doi: 10.1128/mcb.6.6.2233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Deppert W., Haug M., Steinmayer T. Modulation of p53 protein expression during cellular transformation with simian virus 40. Mol Cell Biol. 1987 Dec;7(12):4453–4463. doi: 10.1128/mcb.7.12.4453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Deppert W., Kurth M., Graessmann M., Graessmann A., Knippschild U. Altered phosphorylation at specific sites confers a mutant phenotype to SV40 wild-type large T antigen in a flat revertant of SV40-transformed cells. Oncogene. 1991 Oct;6(10):1931–1938. [PubMed] [Google Scholar]
  10. Deppert W., Steinmayer T., Richter W. Cooperation of SV40 large T antigen and the cellular protein p53 in maintenance of cell transformation. Oncogene. 1989 Sep;4(9):1103–1110. [PubMed] [Google Scholar]
  11. Eliyahu D., Goldfinger N., Pinhasi-Kimhi O., Shaulsky G., Skurnik Y., Arai N., Rotter V., Oren M. Meth A fibrosarcoma cells express two transforming mutant p53 species. Oncogene. 1988 Sep;3(3):313–321. [PubMed] [Google Scholar]
  12. Freeman A. E., Igel H. J., Price P. J. I. In vitrol transformation of rat embryo cells: correlations with the known tumorigenic activities of chemicals in rodents. In Vitro. 1975 Mar-Apr;11(2):107–116. doi: 10.1007/BF02624083. [DOI] [PubMed] [Google Scholar]
  13. Grässer F. A., Scheidtmann K. H., Tuazon P. T., Traugh J. A., Walter G. In vitro phosphorylation of SV40 large T antigen. Virology. 1988 Jul;165(1):13–22. doi: 10.1016/0042-6822(88)90653-8. [DOI] [PubMed] [Google Scholar]
  14. Gurney E. G., Harrison R. O., Fenno J. Monoclonal antibodies against simian virus 40 T antigens: evidence for distinct sublcasses of large T antigen and for similarities among nonviral T antigens. J Virol. 1980 Jun;34(3):752–763. doi: 10.1128/jvi.34.3.752-763.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gurney E. G., Tamowski S., Deppert W. Antigenic binding sites of monoclonal antibodies specific for simian virus 40 large T antigen. J Virol. 1986 Mar;57(3):1168–1172. doi: 10.1128/jvi.57.3.1168-1172.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Harlow E., Crawford L. V., Pim D. C., Williamson N. M. Monoclonal antibodies specific for simian virus 40 tumor antigens. J Virol. 1981 Sep;39(3):861–869. doi: 10.1128/jvi.39.3.861-869.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Herrmann C. P., Kraiss S., Montenarh M. Association of casein kinase II with immunopurified p53. Oncogene. 1991 May;6(5):877–884. [PubMed] [Google Scholar]
  19. Hicks G. G., Egan S. E., Greenberg A. H., Mowat M. Mutant p53 tumor suppressor alleles release ras-induced cell cycle growth arrest. Mol Cell Biol. 1991 Mar;11(3):1344–1352. doi: 10.1128/mcb.11.3.1344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hinzpeter M., Deppert W. Analysis of biological and biochemical parameters for chromatin and nuclear matrix association of SV40 large T antigen in transformed cells. Oncogene. 1987 May;1(2):119–129. [PubMed] [Google Scholar]
  21. Knippschild U., Kiefer J., Patschinsky T., Deppert W. Phenotype-specific phosphorylation of simian virus 40 tsA mutant large T antigens in tsA N-type and A-type transformants. J Virol. 1991 Aug;65(8):4414–4423. doi: 10.1128/jvi.65.8.4414-4423.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Krebs E. G., Beavo J. A. Phosphorylation-dephosphorylation of enzymes. Annu Rev Biochem. 1979;48:923–959. doi: 10.1146/annurev.bi.48.070179.004423. [DOI] [PubMed] [Google Scholar]
  23. Lees-Miller S. P., Chen Y. R., Anderson C. W. Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen. Mol Cell Biol. 1990 Dec;10(12):6472–6481. doi: 10.1128/mcb.10.12.6472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Levine A. J., Momand J. Tumor suppressor genes: the p53 and retinoblastoma sensitivity genes and gene products. Biochim Biophys Acta. 1990 Jun 1;1032(1):119–136. doi: 10.1016/0304-419x(90)90015-s. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Lin J. Y., Simmons D. T. Transformation by simian virus 40 does not involve the mutational activation of p53 to an oncogenic form. Virology. 1990 May;176(1):302–305. doi: 10.1016/0042-6822(90)90258-s. [DOI] [PubMed] [Google Scholar]
  28. Luo K., Hurley T. R., Sefton B. M. Transfer of proteins to membranes facilitates both cyanogen bromide cleavage and two-dimensional proteolytic mapping. Oncogene. 1990 Jun;5(6):921–923. [PubMed] [Google Scholar]
  29. McVey D., Brizuela L., Mohr I., Marshak D. R., Gluzman Y., Beach D. Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication. Nature. 1989 Oct 12;341(6242):503–507. doi: 10.1038/341503a0. [DOI] [PubMed] [Google Scholar]
  30. Meek D. W., Eckhart W. Phosphorylation of p53 in normal and simian virus 40-transformed NIH 3T3 cells. Mol Cell Biol. 1988 Jan;8(1):461–465. doi: 10.1128/mcb.8.1.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Meek D. W., Simon S., Kikkawa U., Eckhart W. The p53 tumour suppressor protein is phosphorylated at serine 389 by casein kinase II. EMBO J. 1990 Oct;9(10):3253–3260. doi: 10.1002/j.1460-2075.1990.tb07524.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Milner J., Cook A., Mason J. p53 is associated with p34cdc2 in transformed cells. EMBO J. 1990 Sep;9(9):2885–2889. doi: 10.1002/j.1460-2075.1990.tb07478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Oren M. The p53 cellular tumor antigen: gene structure, expression and protein properties. Biochim Biophys Acta. 1985 Nov 12;823(1):67–78. doi: 10.1016/0304-419x(85)90015-0. [DOI] [PubMed] [Google Scholar]
  35. Patschinsky T., Bister K. Structural analysis of normal and transforming mil(raf) proteins: effect of 5'-truncation on phosphorylation in vivo or in vitro. Oncogene. 1988 Oct;3(4):357–364. [PubMed] [Google Scholar]
  36. Patschinsky T., Deppert W. Phosphorylation of p53 in primary, immortalised and transformed Balb/c mouse cells. Oncogene. 1990 Jul;5(7):1071–1076. [PubMed] [Google Scholar]
  37. Peden K. W., Srinivasan A., Farber J. M., Pipas J. M. Mutants with changes within or near a hydrophobic region of simian virus 40 large tumor antigen are defective for binding cellular protein p53. Virology. 1989 Jan;168(1):13–21. doi: 10.1016/0042-6822(89)90398-x. [DOI] [PubMed] [Google Scholar]
  38. Pennica D., Goeddel D. V., Hayflick J. S., Reich N. C., Anderson C. W., Levine A. J. The amino acid sequence of murine p53 determined from a c-DNA clone. Virology. 1984 Apr 30;134(2):477–482. doi: 10.1016/0042-6822(84)90316-7. [DOI] [PubMed] [Google Scholar]
  39. Pintel D., Bouck N., di Mayorca G. Separation of lytic and transforming functions of the simian virus 40 A region: two mutants which are temperature sensitive for lytic functions have opposite effects on transformation. J Virol. 1981 May;38(2):518–528. doi: 10.1128/jvi.38.2.518-528.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Richter W., Deppert W. The cellular chromatin is an important target for SV40 large T antigen in maintaining the transformed phenotype. Virology. 1990 Feb;174(2):543–556. doi: 10.1016/0042-6822(90)90108-4. [DOI] [PubMed] [Google Scholar]
  41. Rotter V., Wolf D. Biological and molecular analysis of p53 cellular-encoded tumor antigen. Adv Cancer Res. 1985;43:113–141. doi: 10.1016/s0065-230x(08)60944-6. [DOI] [PubMed] [Google Scholar]
  42. Samad A., Anderson C. W., Carroll R. B. Mapping of phosphomonoester and apparent phosphodiester bonds of the oncogene product p53 from simian virus 40-transformed 3T3 cells. Proc Natl Acad Sci U S A. 1986 Feb;83(4):897–901. doi: 10.1073/pnas.83.4.897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Samad A., Carroll R. B. The tumor suppressor p53 is bound to RNA by a stable covalent linkage. Mol Cell Biol. 1991 Mar;11(3):1598–1606. doi: 10.1128/mcb.11.3.1598. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Scheidtmann K. H., Echle B., Walter G. Simian virus 40 large T antigen is phosphorylated at multiple sites clustered in two separate regions. J Virol. 1982 Oct;44(1):116–133. doi: 10.1128/jvi.44.1.116-133.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Scheidtmann K. H., Haber A. Simian virus 40 large T antigen induces or activates a protein kinase which phosphorylates the transformation-associated protein p53. J Virol. 1990 Feb;64(2):672–679. doi: 10.1128/jvi.64.2.672-679.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Shaulsky G., Goldfinger N., Ben-Ze'ev A., Rotter V. Nuclear accumulation of p53 protein is mediated by several nuclear localization signals and plays a role in tumorigenesis. Mol Cell Biol. 1990 Dec;10(12):6565–6577. doi: 10.1128/mcb.10.12.6565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Soussi T., Caron de Fromentel C., Breugnot C., May E. Nucleotide sequence of a cDNA encoding the rat p53 nuclear oncoprotein. Nucleic Acids Res. 1988 Dec 9;16(23):11384–11384. doi: 10.1093/nar/16.23.11384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Soussi T., Caron de Fromentel C., May P. Structural aspects of the p53 protein in relation to gene evolution. Oncogene. 1990 Jul;5(7):945–952. [PubMed] [Google Scholar]
  49. Steinmeyer K., Maacke H., Deppert W. Cell cycle control by p53 in normal (3T3) and chemically transformed (Meth A) mouse cells. I. Regulation of p53 expression. Oncogene. 1990 Nov;5(11):1691–1699. [PubMed] [Google Scholar]
  50. Stürzbecher H. W., Maimets T., Chumakov P., Brain R., Addison C., Simanis V., Rudge K., Philp R., Grimaldi M., Court W. p53 interacts with p34cdc2 in mammalian cells: implications for cell cycle control and oncogenesis. Oncogene. 1990 Jun;5(6):795–781. [PubMed] [Google Scholar]
  51. Stürzbecher H. W., Montenarh M., Henning R. Enhanced protein phosphorylation in SV40-transformed and -infected cells. Virology. 1987 Oct;160(2):445–455. doi: 10.1016/0042-6822(87)90016-x. [DOI] [PubMed] [Google Scholar]
  52. Tonks N. K., Charbonneau H. Protein tyrosine dephosphorylation and signal transduction. Trends Biochem Sci. 1989 Dec;14(12):497–500. doi: 10.1016/0968-0004(89)90184-9. [DOI] [PubMed] [Google Scholar]
  53. Weber M. J., Edlin G. Phosphate transport, nucleotide pools, and ribonucleic acid synthesis in growing and in density-inhibited 3T3 cells. J Biol Chem. 1971 Mar 25;246(6):1828–1833. [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES