1887

Abstract

Human papillomavirus (HPV) 16 E6 induces the degradation of the tumour suppressor protein p53 by the ubiquitin-dependent proteolysis pathway. , this process involves the formation of a trimolecular complex between E6, p53 and a cellular protein E6-associated protein (E6-AP). However, an analysis of their potential interactions has not been carried out. We have established a model for the expression and analysis of the interactions of these three proteins in insect cells, a eukaryotic system where potentially crucial modifications of the proteins will occur. In baculovirus-infected cells the degradation of p53 can occur. However, p53 is only degraded early in the infectious cycle due to a lack of ATP at later times. Consequently, substantial quantities of material can be produced in this system for further analysis. Evidence is also provided that, , E6 can interact with p53 in the absence of E6-AP and that E6-AP can interact with p53 in the absence of E6. Furthermore, analysis of the subcellular localization of the proteins using both biochemical fractionation and indirect immunofluorescence suggests that the degradation of p53 occurs in the perinuclear region of the cell.

Loading

Article metrics loading...

/content/journal/jgv/10.1099/0022-1317-79-3-489
1998-03-01
2024-12-05
Loading full text...

Full text loading...

/deliver/fulltext/jgv/79/3/9519827.html?itemId=/content/journal/jgv/10.1099/0022-1317-79-3-489&mimeType=html&fmt=ahah

References

  1. Amsterdam A., Pitzer F., Baumeister W. 1993; Changes in intracellular localization of proteasomes in immortalized ovarian granulosa cells during mitosis associated with a role in cell cycle control. Proceedings of the National Academy of Sciences, USA 90:99–103
    [Google Scholar]
  2. Beer-Romero P., Glass S., Rolfe M. 1997; Antisense targeting of E6AP elevates p53 in HPV-infected cells but not in normal cells. Oncogene 14:595–602
    [Google Scholar]
  3. Butel J. S., Jarvis D. L. 1986; The plasma-membrane-associated form of SV40 large tumor antigen: biochemical and biological properties. Biochimica et Biophysica Acta 865:171–195
    [Google Scholar]
  4. Chen J. J., Reid C. E., Band V., Androphy E. J. 1995; Interactions of papillomavirus E6 oncoproteins with a putative calcium-binding protein. Nature 269:529–531
    [Google Scholar]
  5. Chowdary D. R., Dermody J. J., Jha K. K., Ozer H. L. 1994; Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway. Molecular and Cellular Biology 14:1997–2003
    [Google Scholar]
  6. Clarke A. R., Purdie C. A., Harrison D. J., Morris R. G., Bird C. C., Hooper M. L., Wyllie A. H. 1993; Thymocyte apoptosis induced by p53-dependent and independent pathways. Nature 362:849–852
    [Google Scholar]
  7. Daniels P. R., Sanders C. M., Coulson P., Maitland N. J. 1997; Molecular analysis of the interaction between HPV type 16 E6 and human E6-associated protein. FEBS Letters 416:6–10
    [Google Scholar]
  8. Grenfell S. J., Trausch-Azar J. S., Handley-Gearhart P. M., Ciechan-over A., Schwartz A. L. 1994; Nuclear localization of the ubiquitin- activating enzyme, E1, is cell-cycle-dependent. Biochemical Journal 300:701–708
    [Google Scholar]
  9. Grossman S. R., Mora R., Laimins L. A. 1989; Intracellular localization and DNA-binding properties of human papillomavirus type 18 E6 protein expressed with a baculovirus vector. Journal of Virology 63:366–374
    [Google Scholar]
  10. Guarino L. A. 1990; Identification of a viral gene encoding a ubiquitin- like protein. Proceedings of the National Academy of Sciences, USA 87:409–413
    [Google Scholar]
  11. Guarino L. A., Smith G., Dong W. 1995; Ubiquitin is attached to membranes of baculovirus particles by a novel type of phospholipid anchor. Cell 80:301–309
    [Google Scholar]
  12. Guiot M.-C. P., Cavenee W. K., Banks L., Crawford L., Arseneau J., Matlashewski G. 1989; Immunological detection of E6 region protein from human papillomavirus types 16 and 18 in premalignant cervical lesions. Cancer Cells 7:193–196
    [Google Scholar]
  13. Haas A. L., Katzung D. J., Reback P. M., Guarino L. A. 1996; Functional characterization of the ubiquitin variant encoded by the baculovirus Autographa californica . Biochemistry 35:5385–5394
    [Google Scholar]
  14. Hawley-Nelson P., Vousden K. H., Hubbert N. L., Lowy D. R., Schiller J. T. 1989; HPV 16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO Journal 8:3905–3910
    [Google Scholar]
  15. Hershko A. 1991; The ubiquitin pathway for protein degradation. Trends in Biochemical Sciences 16:265–268
    [Google Scholar]
  16. Huibregtse J. M., Scheffner M., Howley P. M. 1991; A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18. EMBO Journal 10:4129–4135
    [Google Scholar]
  17. Huibregtse J. M., Scheffner M., Howley P. M. 1993; Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Molecular and Cellular Biology 13:775–784
    [Google Scholar]
  18. Huibregtse J. M., Scheffner M., Beaudenon S., Howley P. M. 1995; A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proceedings of the National Academy of Sciences, USA 92:2563–2567
    [Google Scholar]
  19. Jentsch S., Seufert W., Hauser H.-P. 1991; Genetic analysis of the ubiquitin system. Biochimica et Biophysica Acta 1089:127–139
    [Google Scholar]
  20. Kanda T., Watanabe S., Zanma S., Sato H., Furuno A., Yoshiike K. 1991; Human papillomavirus type 16 E6 proteins with glycine substitution for cysteine in the metal-binding motif. Virology 185:536–543
    [Google Scholar]
  21. Kastan M. B., Onyekwere O., Sidransky D., Vogelstein B., Craig R. W. 1991; Participation of p53 protein in the cellular response to DNA damage. Cancer Research 51:6304–6311
    [Google Scholar]
  22. Lane D. P. 1992; p53, guardian of the genome. Nature 358:15–16
    [Google Scholar]
  23. Lehman T. A., Modali R., Boukamp P., Stanek J., Bennett W. P., Welsh J. A., Metcalf R. A., Stampfer M. R., Fusenig N., Rogan E. M., Harris C. C. 1993; p53 mutations in human immortalized epithelial cell lines. Carcinogenesis 14:833–839
    [Google Scholar]
  24. Li X., Coffino P. 1996; High -risk human papillomavirus E6 protein has two distinct binding sites within p53, of which only one determines degradation. Journal of Virology 70:4509–4516
    [Google Scholar]
  25. Liang X. H., Volkmann M., Klein R., Herman B., Lockett S. J. 1993; Co-localization of the tumor-suppressor protein p53 and human papillomavirus E6 protein in human cervical carcinoma cell lines. Oncogene 8:2645–2652
    [Google Scholar]
  26. Lowe S. W., Schmitt E. M., Smith S. W., Osborne B. A., Jacks T. 1993; p53 is required for radiation-induced apoptosis in mouse thymocytes. Nature 362:847–849
    [Google Scholar]
  27. Maki C. G., Howley P. M. 1997; Ubiquitination of p53 and p21 is differentially affected by ionizing and UV radiation. Molecular and Cellular Biology 17:355–363
    [Google Scholar]
  28. Maki C. G., Huibregtse J. M., Howley P. M. 1996; In vivo ubiquitination and proteasome-mediated degradation of p53. Cancer Research 56:2649–2654
    [Google Scholar]
  29. Martinez J., Georgoff I., Martinez J., Levine A. J. 1991; Cellular localization and cell cycle regulation by a temperature-sensitive p53 protein. Genes and Development 5:151–159
    [Google Scholar]
  30. Münger K., Phelps W. C., Bubb V., Howley P. M., Schlegel R. 1989; The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. Journal of Virology 63:4417–4421
    [Google Scholar]
  31. O’Reilly D. R., Miller L. K. 1988; Expression and complex formation of simian virus 40 large T antigen and mouse p53 in insect cells. Journal of Virology 62:3109–3119
    [Google Scholar]
  32. Paine P. L., Moore L. C., Horowitz S. B. 1975; Nuclear envelope permeability. Nature 254:109–114
    [Google Scholar]
  33. Rolfe M., Beer-Romero P., Glass S., Eckstein J., Berdo I., Theodoras A., Pagano M., Draetta G. 1995; Reconstitution of p53- ubiquitinylation reactions from purified components: The role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6-AP). Proceedings of the National Academy of Sciences, USA 92:3264–3268
    [Google Scholar]
  34. Sanders C. M., Stern P. L., Maitland N. J. 1995; Characterization of human papillomavirus type 16 E2 protein and subdomains expressed in insect cells. Virology 211:418–433
    [Google Scholar]
  35. Sato H., Watanabe S., Furuno A., Yoshiike K. 1989; Human papillomavirus type 16 E7 protein expressed in Escherichia coli and monkey COS-1 cells: immunofluorescence detection of the nuclear E7 protein. Virology 170:311–315
    [Google Scholar]
  36. Scheffner M., Werness B. A., Huibregtse J. M., Levine A. J., Howley P. M. 1990; The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63:1129–1136
    [Google Scholar]
  37. Scheffner M., Huibregtse J. M., Vierstra R. D., Howley P. M. 1993; The HPV-16 E6 and E6-AP complex functions as a ubiquitin- protein ligase in the ubiquitination of p53. Cell 75:495–505
    [Google Scholar]
  38. Scheffner M., Nuber U., Huibregtse J. M. 1995; Protein ubiquitin- ation involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 373:81–83
    [Google Scholar]
  39. Shaulsky G., Ben-Ze’ev A., Rotter V. 1990; Subcellular distribution of the p53 protein during the cycle of Balb/c 3T3 cells. Oncogene 5:1707–1711
    [Google Scholar]
  40. Shaulsky G., Goldfinger N., Tosky M. S., Levine A. J., Rotter V. 1991; Nuclear localization is essential for the activity of p53 protein. Oncogene 6:2055–2065
    [Google Scholar]
  41. Smotkin D., Wettstein F. O. 1986; Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer- derived cell line and identification of the E7 protein. Proceedings of the National Academy of Sciences, USA 83:4680–4684
    [Google Scholar]
  42. Werness B. A., Levine A. J., Howley P. M. 1990; Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248:76–79
    [Google Scholar]
  43. Wójcik C., Schroeter D., Wilk S., Lamprecht J., Paweletz N. 1996; Ubiquitin-mediated proteolysis centers in HeLa cells: indication from studies of an inhibitor of the chymotrypsin-like activity of the proteasome. European Journal of Cell Biology 71:311–318
    [Google Scholar]
  44. Yonish-Rouach E., Resnitzky D., Lotem J., Sachs L., Kimchi A., Oren M. 1991; Wild-type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin-6. Nature 352:345–347
    [Google Scholar]
  45. Zhan Q., Carrier F., Fornace A. J. Jr 1993; Induction of cellular p53 activity by DNA-damaging agents and growth arrest. Molecular and Cellular Biology 13:4242–4250
    [Google Scholar]
  46. zur Hausen H. 1991; Viruses in human cancers. Science 254:1167–1173
    [Google Scholar]
  47. zur Hausen H., de Villiers E.-M. 1994; Human papillomaviruses. Annual Review of Microbiology 48:427–447
    [Google Scholar]
/content/journal/jgv/10.1099/0022-1317-79-3-489
Loading
/content/journal/jgv/10.1099/0022-1317-79-3-489
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error