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Ubiquitin over-expression promotes E6AP autodegradation and reactivation of the p53/MDM2 pathway in HeLa cells

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Abstract

It has been established that intracellular ubiquitin pools are subject to regulatory constrains. Less certain is the mechanism by which the pool of conjugated ubiquitin shift in parallel with total ubiquitin, and how this type of regulation affects the flux of substrates through the pathway. In this study we demonstrate that ubiquitin over-expression promotes the destabilization of the ubiquitin protein ligase E6AP, by a mechanism involving self-ubiquitination, and the stabilization of p53. These results represent the very first evidence that the levels of a ubiquitin ligase can be regulated in vivo by ubiquitin abundance, supporting the idea that a strict interrelationship between pathway component activities and ubiquitin pool size exists. Interestingly, ubiquitin-induced p53 accumulation did not induce cell-cycle arrest, suggesting that although fluctuations of the intracellular ubiquitin content may actively modulate the level of regulatory proteins, this event is not per se sufficient to elicit a cellular response in terms of proliferation.

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References

  1. Kornitzer D, Ciechanover A (2000) Modes of regulation of ubiquitin-mediated protein degradation. J Cell Physiol 182:1–11. doi :10.1002/(SICI)1097-4652(200001)182:1<1::AID-JCP1>3.0.CO;2-V

    Article  PubMed  CAS  Google Scholar 

  2. Haas AL, Bright PM (1987) The dynamics of ubiquitin pools within cultured human lung fibroblasts. J Biol Chem 262:345–351

    PubMed  CAS  Google Scholar 

  3. Finley D, Ozkaynak E, Varshavsky A (1987) The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell 48:1035–1046. doi:10.1016/0092-8674(87)90711-2

    Article  PubMed  CAS  Google Scholar 

  4. Patel MB, Majetschak M (2007) Distribution and interrelationship of ubiquitin proteasome pathway component activities and ubiquitin pools in various porcine tissues. Physiol Res 56:341–350

    PubMed  CAS  Google Scholar 

  5. Passmore LA, Barford D (2004) Getting into position: the catalytic mechanisms of protein ubiquitylation. Biochem J 379:513–525. doi:10.1042/BJ20040198

    Article  PubMed  CAS  Google Scholar 

  6. Ardley HC, Robinson PA (2005) E3 ubiquitin ligases. Essays Biochem 41:15–30. doi:10.1042/EB0410015

    Article  PubMed  CAS  Google Scholar 

  7. Ang XL, Wade Harper J (2005) SCF-mediated protein degradation and cell cycle control. Oncogene 24:2860–2870. doi:10.1038/sj.onc.1208614

    Article  PubMed  CAS  Google Scholar 

  8. Ryan PE, Davies GC, Nau MM et al (2006) Regulating the regulator: negative regulation of Cbl ubiquitin ligases. Trends Biochem Sci 31:79–88. doi:10.1016/j.tibs.2005.12.004

    Article  PubMed  CAS  Google Scholar 

  9. Nuber U, Schwarz SE, Scheffner M (1998) The ubiquitin-protein ligase E6-associated protein (E6-AP) serves as its own substrate. Eur J Biochem 254:643–649. doi:10.1046/j.1432-1327.1998.2540643.x

    Article  PubMed  CAS  Google Scholar 

  10. Scheffner M, Nuber U, Huibregtse JM (1995) Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 373:81–83. doi:10.1038/373081a0

    Article  PubMed  CAS  Google Scholar 

  11. Huibregtse JM, Scheffner M, Beaudenon S et al (1995) A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc Natl Acad Sci USA 92:2563–2567. doi:10.1073/pnas.92.7.2563

    Article  PubMed  CAS  Google Scholar 

  12. Scheffner M, Werness BA, Huibregtse JM et al (1990) The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63:1129–1136. doi:10.1016/0092-8674(90)90409-8

    Article  PubMed  CAS  Google Scholar 

  13. Scheffner M, Staub O (2007) HECT E3s and human disease. BMC Biochem 8:S6. doi:10.1186/1471-2091-8-S1-S6

    Article  PubMed  Google Scholar 

  14. Kao WH, Beaudenon SL, Talis AL et al (2000) Human papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligase. J Virol 74:6408–6417. doi:10.1128/JVI.74.14.6408-6417.2000

    Article  PubMed  CAS  Google Scholar 

  15. Talis AL, Huibregtse JM, Howley PM (1998) The role of E6-AP in the regulation of p53 protein levels in human papillomavirus (HPV)-positive and HPV-negative cells. J Biol Chem 273:6439–6445. doi:10.1074/jbc.273.11.6439

    Article  PubMed  CAS  Google Scholar 

  16. Lowry OH, Rosebrough NJ, Farr AL et al (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  CAS  Google Scholar 

  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. doi:10.1038/227680a0

    Article  PubMed  CAS  Google Scholar 

  18. Towbin H, Stachelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrilamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354. doi:10.1073/pnas.76.9.4350

    Article  PubMed  CAS  Google Scholar 

  19. Haas AL, Bright PM (1985) The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. J Biol Chem 260:12464–12473

    PubMed  CAS  Google Scholar 

  20. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408. doi:10.1006/meth.2001.1262

    Article  PubMed  CAS  Google Scholar 

  21. Callis J, Ling R (2005) Epitope-tagged ubiquitin. A new probe for analyzing ubiquitin function. Methods Enzymol 399:51–64. doi:10.1016/S0076-6879(05)99004-6

    Article  PubMed  CAS  Google Scholar 

  22. Finley D, Bartel B, Varshavsky A (1989) The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature 338:394–401. doi:10.1038/338394a0

    Article  PubMed  CAS  Google Scholar 

  23. Ozkaynak E, Finley D, Solomon MJ, Varshavsky A (1987) The yeast ubiquitin genes: a family of natural gene fusions. EMBO J 6:1429–1439

    PubMed  CAS  Google Scholar 

  24. Tsirigotis M, Zhang M, Chiu RK et al (2001) Sensitivity of mammalian cells expressing mutant ubiquitin to protein-damaging agents. J Biol Chem 276:46073–46078. doi:10.1074/jbc.M109023200

    Article  PubMed  CAS  Google Scholar 

  25. Ryu KY, Baker RT, Kopito RR (2006) Ubiquitin-specific protease 2 as a tool for quantification of total ubiquitin levels in biological specimens. Anal Biochem 353:153–155. doi:10.1016/j.ab.2006.03.038

    Article  PubMed  CAS  Google Scholar 

  26. Igoucheva O, Alexeev V, Yoon K (2006) Differential cellular responses to exogenous DNA in mammalian cells and its effect on oligonucleotide-directed gene modification. Gene Ther 13:266–275. doi:10.1038/sj.gt.3302643

    Article  PubMed  CAS  Google Scholar 

  27. Wang M, Pickart CM (2005) Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis. EMBO J 24:4324–4333. doi:10.1038/sj.emboj.7600895

    Article  PubMed  CAS  Google Scholar 

  28. Laine A, Topisirovic I, Zhai D et al (2006) Regulation of p53 localization and activity by Ubc13. Mol Cell Biol 26:8901–8913. doi:10.1128/MCB.01156-06

    Article  PubMed  CAS  Google Scholar 

  29. Kim HT, Kim KP, Lledias F et al (2007) Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages. J Biol Chem 282:17375–17386. doi:10.1074/jbc.M609659200

    Article  PubMed  CAS  Google Scholar 

  30. Kovalenko A, Wallach D (2006) If the prophet does not come to the mountain: dynamics of signaling complexes in NF-kappaB activation. Mol Cell 22:433–436. doi:10.1016/j.molcel.2006.05.002

    Article  PubMed  CAS  Google Scholar 

  31. Appella E, Anderson CW (2001) Post-translational modifications and activation of p53 by genotoxic stresses. Eur J Biochem 268:2764–2772. doi:10.1046/j.1432-1327.2001.02225.x

    Article  PubMed  CAS  Google Scholar 

  32. Sarkaria JN, Tibbetts RS, Busby EC et al (1998) Inhibition of phosphoinositide 3-kinase related kinases by the radiosensitizing agent wortmannin. Cancer Res 58:4375–4382

    PubMed  CAS  Google Scholar 

  33. Goodwin EC, DiMaio D (2000) Repression of human papillomavirus oncogenes in HeLa cervical carcinoma cells causes the orderly reactivation of dormant tumor suppressor pathways. Proc Natl Acad Sci USA 97:12513–12518. doi:10.1073/pnas.97.23.12513

    Article  PubMed  CAS  Google Scholar 

  34. Cheng TH, Cohen SN (2007) Human MDM2 isoforms translated differentially on constitutive versus p53-regulated transcripts have distinct functions in the p53/MDM2 and TSG101/MDM2 feedback control loops. Mol Cell Biol 27:111–119. doi:10.1128/MCB.00235-06

    Article  PubMed  CAS  Google Scholar 

  35. Mao X, Stewart AK, Hurren R et al (2007) A chemical biology screen identifies glucocorticoids that regulate c-maf expression by increasing its proteasomal degradation through upregulation of ubiquitin. Blood 110:4047–4054. doi:10.1182/blood-2007-05-088666

    Article  PubMed  CAS  Google Scholar 

  36. Daino H, Matsumura I, Takada K et al (2000) Induction of apoptosis by extracellular ubiquitin in human hematopoietic cells: possible involvement of STAT3 degradation by proteasome pathway in interleukin 6-dependent hematopoietic cells. Blood 95:2577–2585

    PubMed  CAS  Google Scholar 

  37. Kutty BC, Pasupathy K, Mishra KP (2005) Effects of exogenous ubiquitin on cell division cycle mutants of Schizosaccharomyces pombe. FEMS Microbiol Lett 244:187–191. doi:10.1016/j.femsle.2005.01.044

    Article  PubMed  CAS  Google Scholar 

  38. Kee Y, Huibregtse JM (2007) Regulation of catalytic activities of HECT ubiquitin ligases. Biochem Biophys Res Commun 354:329–333. doi:10.1016/j.bbrc.2007.01.025

    Article  PubMed  CAS  Google Scholar 

  39. Siepmann TJ, Bohnsack RN, Tokgoz Z et al (2003) Protein interactions within the N-end rule ubiquitin ligation pathway. J Biol Chem 278:9448–9457. doi:10.1074/jbc.M211240200

    Article  PubMed  CAS  Google Scholar 

  40. Dantuma NP, Groothuis TA, Salomons FA et al (2006) A dynamic ubiquitin equilibrium couples proteasomal activity to chromatin remodeling. J Cell Biol 173:19–26. doi:10.1083/jcb.200510071

    Article  PubMed  CAS  Google Scholar 

  41. Camus S, Menéndez S, Cheok CF et al (2007) Ubiquitin-independent degradation of p53 mediated by high-risk human papillomavirus protein E6. Oncogene 26:4059–4070. doi:10.1038/sj.onc.1210188

    Article  PubMed  CAS  Google Scholar 

  42. Massimi P, Shai A, Lambert P et al (2007) HPV E6 degradation of p53 and PDZ containing substrates in an E6AP null background. Oncogene 27:1800–1804

    Article  PubMed  Google Scholar 

  43. 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. doi:10.1038/sj.onc.1200872

    Article  PubMed  CAS  Google Scholar 

  44. Kim Y, Cairns MJ, Marouga R et al (2003) E6AP gene suppression and characterization with in vitro selected hammerhead ribozymes. Cancer Gene Ther 10:707–716. doi:10.1038/sj.cgt.7700623

    Article  PubMed  CAS  Google Scholar 

  45. Hengstermann A, D’silva MA, Kuballa P et al (2005) Growth suppression induced by downregulation of E6-AP expression in human papillomavirus-positive cancer cell lines depends on p53. J Virol 79:9296–9300. doi:10.1128/JVI.79.14.9296-9300.2005

    Article  PubMed  CAS  Google Scholar 

  46. Kelley ML, Keiger KE, Lee CJ et al (2005) The global transcriptional effects of the human papillomavirus E6 protein in cervical carcinoma cell lines are mediated by the E6AP ubiquitin ligase. J Virol 79:3737–3747. doi:10.1128/JVI.79.6.3737-3747.2005

    Article  PubMed  CAS  Google Scholar 

  47. Oliner JD, Pietenpol JA, Thiagalingam S et al (1993) Oncoprotein MDM2 conceals the activation domain of tumour suppressor p53. Nature 362:857–860. doi:10.1038/362857a0

    Article  PubMed  CAS  Google Scholar 

  48. Haupt Y, Maya R, Kazaz A et al (1997) Mdm2 promotes the rapid degradation of p53. Nature 387:296–299. doi:10.1038/387296a0

    Article  PubMed  CAS  Google Scholar 

  49. Zhang Z, Wang H, Li M et al (2004) MDM2 is a negative regulator of p21WAF1/CIP1, independent of p53. J Biol Chem 279:16000–16006. doi:10.1074/jbc.M312264200

    Article  PubMed  CAS  Google Scholar 

  50. Stommel JM, Wahl GM (2005) A new twist in the feedback loop: stress-activated MDM2 destabilization is required for p53 activation. Cell Cycle 4:411–417

    PubMed  CAS  Google Scholar 

  51. Hanna J, Meides A, Zhang DP et al (2007) A ubiquitin stress response induces altered proteasome composition. Cell 129:747–759. doi:10.1016/j.cell.2007.03.042

    Article  PubMed  CAS  Google Scholar 

  52. Delic J, Morange M, Magdelenat H (1993) Ubiquitin pathway involvement in human lymphocyte γ-irradiation-induced apoptosis. Mol Cell Biol 13:4875–4883

    PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by COFIN-MIUR PRIN 2003 (prot. 2003058397_001) and 2006 (prot. 2006058482_001) and FIRB (PNR 2001 RBNE01T8C8-008) granted to M. Magnani. The authors would like to thank Prof. A. L. Haas for providing the anti-ubiquitin antibody used in this study. Plasmids encoding E6AP and E6AP C833A were kindly made available by Prof. Peter Howley through Addgene (http://www.addgene.org), a non-profit research support service dedicated to archiving and distributing plasmids that appear in published articles.

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Authors and Affiliations

  1. Istituto di Chimica Biologica “G. Fornaini”, Università degli Studi di Urbino “Carlo Bo”, via Saffi, 2, 61029, Urbino, PU, Italy

    Rita Crinelli, Marzia Bianchi, Michele Menotta, Elisa Carloni, Elisa Giacomini & Mauro Magnani

  2. Dipartimento di Oncologia Sperimentale, Fondazione IRCCS Istituto Nazionale dei Tumori, 20133, Milan, Italy

    Marzia Pennati

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  1. Rita Crinelli
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  2. Marzia Bianchi
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  3. Michele Menotta
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Correspondence to Rita Crinelli.

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Crinelli, R., Bianchi, M., Menotta, M. et al. Ubiquitin over-expression promotes E6AP autodegradation and reactivation of the p53/MDM2 pathway in HeLa cells. Mol Cell Biochem 318, 129–145 (2008). https://doi.org/10.1007/s11010-008-9864-8

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