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. 1994 Sep 13;91(19):8797–8801. doi: 10.1073/pnas.91.19.8797

Identification of a human ubiquitin-conjugating enzyme that mediates the E6-AP-dependent ubiquitination of p53.

M Scheffner 1, J M Huibregtse 1, P M Howley 1
PMCID: PMC44693  PMID: 8090726

Abstract

The E6 protein of the oncogenic human papillomavirus types 16 and 18 facilitates the rapid degradation of the tumor-suppressor protein p53 via the ubiquitin-dependent proteolytic pathway. The E6 protein binds to a cellular protein of 100 kDa termed E6-AP. The complex of E6 and E6-AP specifically interacts with p53 and induces the ubiquitination of p53 in a reaction which requires the ubiquitin-activating enzyme (E1) and a cellular fraction thought to contain a mammalian ubiquitin-conjugating enzyme (E2). This mammalian E2 activity could be replaced with bacterially expressed UBC8 from Arabidopsis thaliana, which belongs to a subfamily of E2s including yeast UBC4 and UBC5 which are highly conserved at the amino acid level. In this paper we describe the cloning of a human cDNA encoding a human E2 that we have designated UbcH5 and that is related to Arabidopsis UBC8 and the other members of this subfamily. We demonstrate that UbcH5 can function in the E6/E6-AP-induced ubiquitination of p53.

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Selected References

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  1. Band V., De Caprio J. A., Delmolino L., Kulesa V., Sager R. Loss of p53 protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells. J Virol. 1991 Dec;65(12):6671–6676. doi: 10.1128/jvi.65.12.6671-6676.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bartel B., Wünning I., Varshavsky A. The recognition component of the N-end rule pathway. EMBO J. 1990 Oct;9(10):3179–3189. doi: 10.1002/j.1460-2075.1990.tb07516.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blumenfeld N., Gonen H., Mayer A., Smith C. E., Siegel N. R., Schwartz A. L., Ciechanover A. Purification and characterization of a novel species of ubiquitin-carrier protein, E2, that is involved in degradation of non-"N-end rule" protein substrates. J Biol Chem. 1994 Apr 1;269(13):9574–9581. [PubMed] [Google Scholar]
  4. Chau V., Tobias J. W., Bachmair A., Marriott D., Ecker D. J., Gonda D. K., Varshavsky A. A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science. 1989 Mar 24;243(4898):1576–1583. doi: 10.1126/science.2538923. [DOI] [PubMed] [Google Scholar]
  5. Chowdary D. R., Dermody J. J., Jha K. K., Ozer H. L. Accumulation of p53 in a mutant cell line defective in the ubiquitin pathway. Mol Cell Biol. 1994 Mar;14(3):1997–2003. doi: 10.1128/mcb.14.3.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ciechanover A., DiGiuseppe J. A., Bercovich B., Orian A., Richter J. D., Schwartz A. L., Brodeur G. M. Degradation of nuclear oncoproteins by the ubiquitin system in vitro. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):139–143. doi: 10.1073/pnas.88.1.139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ciechanover A., Shkedy D., Oren M., Bercovich B. Degradation of the tumor suppressor protein p53 by the ubiquitin-mediated proteolytic system requires a novel species of ubiquitin-carrier protein, E2. J Biol Chem. 1994 Apr 1;269(13):9582–9589. [PubMed] [Google Scholar]
  8. Dohmen R. J., Madura K., Bartel B., Varshavsky A. The N-end rule is mediated by the UBC2(RAD6) ubiquitin-conjugating enzyme. Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7351–7355. doi: 10.1073/pnas.88.16.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Finley D., Chau V. Ubiquitination. Annu Rev Cell Biol. 1991;7:25–69. doi: 10.1146/annurev.cb.07.110191.000325. [DOI] [PubMed] [Google Scholar]
  10. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Girod P. A., Carpenter T. B., van Nocker S., Sullivan M. L., Vierstra R. D. Homologs of the essential ubiquitin conjugating enzymes UBC1, 4, and 5 in yeast are encoded by a multigene family in Arabidopsis thaliana. Plant J. 1993 Apr;3(4):545–552. doi: 10.1046/j.1365-313x.1993.03040545.x. [DOI] [PubMed] [Google Scholar]
  12. Girod P. A., Vierstra R. D. A major ubiquitin conjugation system in wheat germ extracts involves a 15-kDa ubiquitin-conjugating enzyme (E2) homologous to the yeast UBC4/UBC5 gene products. J Biol Chem. 1993 Jan 15;268(2):955–960. [PubMed] [Google Scholar]
  13. Glotzer M., Murray A. W., Kirschner M. W. Cyclin is degraded by the ubiquitin pathway. Nature. 1991 Jan 10;349(6305):132–138. doi: 10.1038/349132a0. [DOI] [PubMed] [Google Scholar]
  14. Goebl M. G., Yochem J., Jentsch S., McGrath J. P., Varshavsky A., Byers B. The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme. Science. 1988 Sep 9;241(4871):1331–1335. doi: 10.1126/science.2842867. [DOI] [PubMed] [Google Scholar]
  15. Haas A. L., Warms J. V., Hershko A., Rose I. A. Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation. J Biol Chem. 1982 Mar 10;257(5):2543–2548. [PubMed] [Google Scholar]
  16. Hatfield P. M., Vierstra R. D. Multiple forms of ubiquitin-activating enzyme E1 from wheat. Identification of an essential cysteine by in vitro mutagenesis. J Biol Chem. 1992 Jul 25;267(21):14799–14803. [PubMed] [Google Scholar]
  17. Hershko A., Ciechanover A. The ubiquitin system for protein degradation. Annu Rev Biochem. 1992;61:761–807. doi: 10.1146/annurev.bi.61.070192.003553. [DOI] [PubMed] [Google Scholar]
  18. Hochstrasser M., Ellison M. J., Chau V., Varshavsky A. The short-lived MAT alpha 2 transcriptional regulator is ubiquitinated in vivo. Proc Natl Acad Sci U S A. 1991 Jun 1;88(11):4606–4610. doi: 10.1073/pnas.88.11.4606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hubbert N. L., Sedman S. A., Schiller J. T. Human papillomavirus type 16 E6 increases the degradation rate of p53 in human keratinocytes. J Virol. 1992 Oct;66(10):6237–6241. doi: 10.1128/jvi.66.10.6237-6241.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Huibregtse J. M., Scheffner M., Howley P. M. A cellular protein mediates association of p53 with the E6 oncoprotein of human papillomavirus types 16 or 18. EMBO J. 1991 Dec;10(13):4129–4135. doi: 10.1002/j.1460-2075.1991.tb04990.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Huibregtse J. M., Scheffner M., Howley P. M. Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papillomavirus E6 oncoprotein with p53. Mol Cell Biol. 1993 Feb;13(2):775–784. doi: 10.1128/mcb.13.2.775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Huibregtse J. M., Scheffner M., Howley P. M. Localization of the E6-AP regions that direct human papillomavirus E6 binding, association with p53, and ubiquitination of associated proteins. Mol Cell Biol. 1993 Aug;13(8):4918–4927. doi: 10.1128/mcb.13.8.4918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jentsch S., McGrath J. P., Varshavsky A. The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme. Nature. 1987 Sep 10;329(6135):131–134. doi: 10.1038/329131a0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Plon S. E., Leppig K. A., Do H. N., Groudine M. Cloning of the human homolog of the CDC34 cell cycle gene by complementation in yeast. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10484–10488. doi: 10.1073/pnas.90.22.10484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Scheffner M., Münger K., Byrne J. C., Howley P. M. The state of the p53 and retinoblastoma genes in human cervical carcinoma cell lines. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5523–5527. doi: 10.1073/pnas.88.13.5523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Scheffner M., Takahashi T., Huibregtse J. M., Minna J. D., Howley P. M. Interaction of the human papillomavirus type 16 E6 oncoprotein with wild-type and mutant human p53 proteins. J Virol. 1992 Aug;66(8):5100–5105. doi: 10.1128/jvi.66.8.5100-5105.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Schneider R., Eckerskorn C., Lottspeich F., Schweiger M. The human ubiquitin carrier protein E2(Mr = 17,000) is homologous to the yeast DNA repair gene RAD6. EMBO J. 1990 May;9(5):1431–1435. doi: 10.1002/j.1460-2075.1990.tb08259.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Seufert W., McGrath J. P., Jentsch S. UBC1 encodes a novel member of an essential subfamily of yeast ubiquitin-conjugating enzymes involved in protein degradation. EMBO J. 1990 Dec;9(13):4535–4541. doi: 10.1002/j.1460-2075.1990.tb07905.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sullivan M. L., Vierstra R. D. Cloning of a 16-kDa ubiquitin carrier protein from wheat and Arabidopsis thaliana. Identification of functional domains by in vitro mutagenesis. J Biol Chem. 1991 Dec 15;266(35):23878–23885. [PubMed] [Google Scholar]
  35. Treier M., Seufert W., Jentsch S. Drosophila UbcD1 encodes a highly conserved ubiquitin-conjugating enzyme involved in selective protein degradation. EMBO J. 1992 Jan;11(1):367–372. doi: 10.1002/j.1460-2075.1992.tb05059.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]

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