Reconstitution of p53-ubiquitinylation reactions from purified components: the role of human ubiquitin-conjugating enzyme UBC4 and E6-associated protein (E6AP) (original) (raw)

Abstract

The E6 protein of the high-risk human papillomaviruses inactivates the tumor suppressor protein p53 by stimulating its ubiquitinylation and subsequent degradation. Ubiquitinylation is a multistep process involving a ubiquitin-activating enzyme, one of many distinct ubiquitin-conjugating enzymes, and in certain cases, a ubiquitin ligase. In human papillomavirus-infected cells, E6 and the E6-associated protein are thought to act as a ubiquitin-protein ligase in the ubiquitinylation of p53. Here we describe the cloning of a human ubiquitin-conjugating enzyme that specifically ubiquitinylates E6-associated protein. Furthermore, we define the biochemical pathway of p53 ubiquitinylation and demonstrate that in vivo inhibition of various components in the pathway leads to an inhibition of E6-stimulated p53 degradation.

3264

Images in this article

Selected References

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

  1. Ansorge W., Pepperkok R. Performance of an automated system for capillary microinjection into living cells. J Biochem Biophys Methods. 1988 Aug;16(4):283–292. doi: 10.1016/0165-022x(88)90062-0. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Dyson N., Howley P. M., Münger K., Harlow E. The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science. 1989 Feb 17;243(4893):934–937. doi: 10.1126/science.2537532. [DOI] [PubMed] [Google Scholar]
  4. Handley P. M., Mueckler M., Siegel N. R., Ciechanover A., Schwartz A. L. Molecular cloning, sequence, and tissue distribution of the human ubiquitin-activating enzyme E1. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):258–262. doi: 10.1073/pnas.88.1.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Jentsch S. The ubiquitin-conjugation system. Annu Rev Genet. 1992;26:179–207. doi: 10.1146/annurev.ge.26.120192.001143. [DOI] [PubMed] [Google Scholar]
  7. Kaiser P., Seufert W., Höfferer L., Kofler B., Sachsenmaier C., Herzog H., Jentsch S., Schweiger M., Schneider R. A human ubiquitin-conjugating enzyme homologous to yeast UBC8. J Biol Chem. 1994 Mar 25;269(12):8797–8802. [PubMed] [Google Scholar]
  8. Koken M. H., Reynolds P., Jaspers-Dekker I., Prakash L., Prakash S., Bootsma D., Hoeijmakers J. H. Structural and functional conservation of two human homologs of the yeast DNA repair gene RAD6. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8865–8869. doi: 10.1073/pnas.88.20.8865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Koken M., Reynolds P., Bootsma D., Hoeijmakers J., Prakash S., Prakash L. Dhr6, a Drosophila homolog of the yeast DNA-repair gene RAD6. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3832–3836. doi: 10.1073/pnas.88.9.3832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Liu Z., Diaz L. A., Haas A. L., Giudice G. J. cDNA cloning of a novel human ubiquitin carrier protein. An antigenic domain specifically recognized by endemic pemphigus foliaceus autoantibodies is encoded in a secondary reading frame of this human epidermal transcript. J Biol Chem. 1992 Aug 5;267(22):15829–15835. [PubMed] [Google Scholar]
  11. Scheffner M., Huibregtse J. M., Howley P. M. Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8797–8801. doi: 10.1073/pnas.91.19.8797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Scheffner M., Huibregtse J. M., Vierstra R. D., Howley P. M. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell. 1993 Nov 5;75(3):495–505. doi: 10.1016/0092-8674(93)90384-3. [DOI] [PubMed] [Google Scholar]
  13. Scheffner M., Nuber U., Huibregtse J. M. Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature. 1995 Jan 5;373(6509):81–83. doi: 10.1038/373081a0. [DOI] [PubMed] [Google Scholar]
  14. Scheffner M., Werness B. A., Huibregtse J. M., Levine A. J., Howley P. M. The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell. 1990 Dec 21;63(6):1129–1136. doi: 10.1016/0092-8674(90)90409-8. [DOI] [PubMed] [Google Scholar]
  15. Seufert W., Jentsch S. Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins. EMBO J. 1990 Feb;9(2):543–550. doi: 10.1002/j.1460-2075.1990.tb08141.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Shaw P., Bovey R., Tardy S., Sahli R., Sordat B., Costa J. Induction of apoptosis by wild-type p53 in a human colon tumor-derived cell line. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4495–4499. doi: 10.1073/pnas.89.10.4495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Superti-Furga G., Bergers G., Picard D., Busslinger M. Hormone-dependent transcriptional regulation and cellular transformation by Fos-steroid receptor fusion proteins. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5114–5118. doi: 10.1073/pnas.88.12.5114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Varshavsky A. The N-end rule. Cell. 1992 May 29;69(5):725–735. doi: 10.1016/0092-8674(92)90285-k. [DOI] [PubMed] [Google Scholar]
  19. Vogelstein B., Kinzler K. W. p53 function and dysfunction. Cell. 1992 Aug 21;70(4):523–526. doi: 10.1016/0092-8674(92)90421-8. [DOI] [PubMed] [Google Scholar]
  20. Vojtesek B., Bártek J., Midgley C. A., Lane D. P. An immunochemical analysis of the human nuclear phosphoprotein p53. New monoclonal antibodies and epitope mapping using recombinant p53. J Immunol Methods. 1992 Jul 6;151(1-2):237–244. doi: 10.1016/0022-1759(92)90122-a. [DOI] [PubMed] [Google Scholar]
  21. Werness B. A., Levine A. J., Howley P. M. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science. 1990 Apr 6;248(4951):76–79. doi: 10.1126/science.2157286. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Zhen M., Heinlein R., Jones D., Jentsch S., Candido E. P. The ubc-2 gene of Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme involved in selective protein degradation. Mol Cell Biol. 1993 Mar;13(3):1371–1377. doi: 10.1128/mcb.13.3.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. zur Hausen H. Viruses in human cancers. Science. 1991 Nov 22;254(5035):1167–1173. doi: 10.1126/science.1659743. [DOI] [PubMed] [Google Scholar]