1887

Journal of General Virology header logo

Volume 97, Issue 12

Ubiquitin ligase E6AP mediates nonproteolytic polyubiquitylation of -catenin independent of the E6 oncoprotein Free

Abstract

Recently, we showed that the ubiquitin ligase E6AP stabilizes β-catenin and activates its transcriptional activity. These activities were enhanced by the human papillomavirus (HPV) E6 protein. In the present study, we explored the function of E6AP, which increases β-catenin stabilization and transcriptional activation. Here, we report that E6AP interacts with β-catenin and mediates its nonproteolytic ubiquitylation, as evidenced in transiently transfected cell-based and reconstitution ubiquitylation assays. Overexpression of E6AP increased β-catenin polyubiquitylation and, consistent with that, knockdown or knock-out of E6AP expression reduced β-catenin polyubiquitylation. The ubiquitylation of β-catenin by E6AP was dependent on its E3 ubiquitin ligase activity, but it was proteasome-independent and did not require HPV-E6, phosphorylation of β-catenin by glycogen synthase kinase 3β (GSK3β) or activity of the β-catenin ‘destruction complex’. We also show that transcriptional activation of β-catenin by E6AP is coupled with β-catenin protein stabilization, but not its ubiquitylation. In contrast to β-catenin ubiquitylation, β-catenin protein stability and its transcriptional activity were absolutely dependent on the activity of the destruction complex and phosphorylation by GSK3β. Collectively, our data uncover a dual role for E6AP in the regulation of β-catenin ubiquitylation, stability and transcriptional activity, with HPV-E6 enhancing only part of E6AP activities.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): E6AP , HPV E6 , ubiquitylation , Wnt signaling and β-catenin
© 2016 The Authors
Loading

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.000624
2016-12-16
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/jgv/97/12/3313.html?itemId=/content/journal/jgv/10.1099/jgv.0.000624&mimeType=html&fmt=ahah

References

  1. Amit S., Hatzubai A., Birman Y., Andersen J. S., Ben-Shushan E., Mann M., Ben-Neriah Y., Alkalay I. 2002; Axin-mediated CKI phosphorylation of β-catenin at Ser 45: a molecular switch for the Wnt pathway. Genes Dev 16:1066–1076 [View Article][PubMed]
     [Google Scholar]
  2. Bello J. O., Nieva L. O., Paredes A. C., Gonzalez A. M., Zavaleta L. R., Lizano M. 2015; Regulation of the Wnt/β-catenin signaling pathway by human papillomavirus E6 and E7 oncoproteins. Viruses 7:4734–4755 [View Article][PubMed]
     [Google Scholar]
  3. Caspi M., Zilberberg A., Eldar-Finkelman H., Rosin-Arbesfeld R. 2008; Nuclear GSK-3β inhibits the canonical Wnt signalling pathway in a β-catenin phosphorylation-independent manner. Oncogene 27:3546–3555 [View Article][PubMed]
     [Google Scholar]
  4. Caspi M., Perry G., Skalka N., Meisel S., Firsow A., Amit M., Rosin-Arbesfeld R. 2014; Aldolase positively regulates of the canonical Wnt signaling pathway. Mol Cancer 13:164 [View Article][PubMed]
     [Google Scholar]
  5. Chitalia V. C., Foy R. L., Bachschmid M. M., Zeng L., Panchenko M. V., Zhou M. I., Bharti A., Seldin D. C., Lecker S. H. et al. 2008; Jade-1 inhibits Wnt signalling by ubiquitylating β-catenin and mediates Wnt pathway inhibition by pVHL. Nat Cell Biol 10:1208–1216 [View Article][PubMed]
     [Google Scholar]
  6. Chitalia V., Shivanna S., Martorell J., Meyer R., Edelman E., Rahimi N. 2013; c-Cbl, a ubiquitin E3 ligase that targets active β-catenin: a novel layer of Wnt signaling regulation. J Biol Chem 288:23505–23517 [View Article][PubMed]
     [Google Scholar]
  7. Clevers H. 2006; Wnt/β-catenin signaling in development and disease. Cell 127:469–480 [View Article][PubMed]
     [Google Scholar]
  8. Clevers H., Nusse R. 2012; Wnt/β-catenin signaling and disease. Cell 149:1192–1205 [View Article][PubMed]
     [Google Scholar]
  9. de Bie P., Zaaroor-Regev D., Ciechanover A. 2010; Regulation of the polycomb protein RING1B ubiquitination by USP7. Biochem Biophys Res Commun 400:389–395 [View Article][PubMed]
     [Google Scholar]
  10. El Hokayem J., Nawaz Z. 2014; E6AP in the brain: one protein, dual function, multiple diseases. Mol Neurobiol 49:827–839 [View Article][PubMed]
     [Google Scholar]
  11. Essers M. A., de Vries-Smits L. M., Barker N., Polderman P. E., Burgering B. M., Korswagen H. C. 2005; Functional interaction between beta-catenin and FOXO in oxidative stress signaling. Science 308:1181–1184 [View Article][PubMed]
     [Google Scholar]
  12. Fagotto F. 2013; Looking beyond the Wnt pathway for the deep nature of β-catenin. EMBO Rep 14:422–433 [View Article][PubMed]
     [Google Scholar]
  13. Fang P., Lev-Lehman E., Tsai T. F., Matsuura T., Benton C. S., Sutcliffe J. S., Christian S. L., Kubota T., Halley D. J. et al. 1999; The spectrum of mutations in UBE3A causing Angelman syndrome. Hum Mol Genet 8:129–135 [View Article][PubMed]
     [Google Scholar]
  14. Fei C., Li Z., Li C., Chen Y., Chen Z., He X., Mao L., Wang X., Zeng R., Li L. 2013; Smurf1-mediated Lys29-linked nonproteolytic polyubiquitination of axin negatively regulates Wnt/β-catenin signaling. Mol Cell Biol 33:4095–4105 [View Article][PubMed]
     [Google Scholar]
  15. Fujita Y., Krause G., Scheffner M., Zechner D., Leddy H. E., Behrens J., Sommer T., Birchmeier W. 2002; Hakai, a c-Cbl-like protein, ubiquitinates and induces endocytosis of the E-cadherin complex. Nat Cell Biol 4:222–231 [View Article][PubMed]
     [Google Scholar]
  16. Gerard B., Sanders M. A., Visscher D. W., Tait L., Shekhar M. P. 2012; Lysine 394 is a novel Rad6B-induced ubiquitination site on beta-catenin. Biochim Biophys Acta 1823:1686–1696 [View Article][PubMed]
     [Google Scholar]
  17. Greer P. L., Hanayama R., Bloodgood B. L., Mardinly A. R., Lipton D. M., Flavell S. W., Kim T. K., Griffith E. C., Waldon Z. et al. 2010; The Angelman syndrome protein Ube3A regulates synapse development by ubiquitinating arc. Cell 140:704–716 [View Article][PubMed]
     [Google Scholar]
  18. Hay-Koren A., Caspi M., Zilberberg A., Rosin-Arbesfeld R. 2011; The EDD E3 ubiquitin ligase ubiquitinates and up-regulates β-catenin. Mol Biol Cell 22:399–411 [View Article][PubMed]
     [Google Scholar]
  19. Howie H. L., Katzenellenbogen R. A., Galloway D. A. 2009; Papillomavirus E6 proteins. Virology 384:324–334 [View Article][PubMed]
     [Google Scholar]
  20. Kimelman D., Xu W. 2006; β-Catenin destruction complex: insights and questions from a structural perspective. Oncogene 25:7482–7491 [View Article][PubMed]
     [Google Scholar]
  21. Komander D., Rape M. 2012; The ubiquitin code. Annu Rev Biochem 81:203–229 [View Article][PubMed]
     [Google Scholar]
  22. Komiya Y., Habas R. 2008; Wnt signal transduction pathways. Organogenesis 4:68–75 [View Article][PubMed]
     [Google Scholar]
  23. Kulathu Y., Komander D. 2012; Atypical ubiquitylation — the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages. Nat Rev Mol Cell Biol 13:508–523 [View Article][PubMed]
     [Google Scholar]
  24. Kumar S., Talis A. L., Howley P. M. 1999; Identification of HHR23A as a substrate for E6-associated protein-mediated ubiquitination. J Biol Chem 274:18785–18792 [View Article][PubMed]
     [Google Scholar]
  25. Levav-Cohen Y., Wolyniec K., Alsheich-Bartok O., Chan A. L., Woods S. J., Jiang Y. H., Haupt S., Haupt Y. 2012; E6AP is required for replicative and oncogene-induced senescence in mouse embryo fibroblasts. Oncogene 31:2199–2209 [View Article][PubMed]
     [Google Scholar]
  26. Lichtig H., Gilboa D. A., Jackman A., Gonen P., Levav-Cohen Y., Haupt Y., Sherman L. 2010; HPV16 E6 augments Wnt signaling in an E6AP-dependent manner. Virology 396:47–58 [View Article][PubMed]
     [Google Scholar]
  27. Liu J., Stevens J., Rote C. A., Yost H. J., Hu Y., Neufeld K. L., White R. L., Matsunami N. 2001; Siah-1 mediates a novel β-catenin degradation pathway linking p53 to the adenomatous polyposis coli protein. Mol Cell 7:927–936 [View Article][PubMed]
     [Google Scholar]
  28. Liu C., Li Y., Semenov M., Han C., Baeg G. H., Tan Y., Zhang Z., Lin X., He X. 2002; Control of β-catenin phosphorylation/degradation by a dual-kinase mechanism. Cell 108:837–847 [View Article][PubMed]
     [Google Scholar]
  29. Louria-Hayon I., Alsheich-Bartok O., Levav-Cohen Y., Silberman I., Berger M., Grossman T., Matentzoglu K., Jiang Y. H., Muller S. et al. 2009; E6AP promotes the degradation of the PML tumor suppressor. Cell Death Differ 16:1156–1166 [View Article][PubMed]
     [Google Scholar]
  30. Martinez-Forero I., Rouzaut A., Palazon A., Dubrot J., Melero I. 2009; Lysine 63 polyubiquitination in immunotherapy and in cancer-promoting inflammation. Clin Cancer Res 15:6751–6757 [View Article][PubMed]
     [Google Scholar]
  31. Martínez-Noël G., Galligan J. T., Sowa M. E., Arndt V., Overton T. M., Harper J. W., Howley P. M. 2012; Identification and proteomic analysis of distinct UBE3A/E6AP protein complexes. Mol Cell Biol 32:3095–3106 [View Article][PubMed]
     [Google Scholar]
  32. Matentzoglu K., Scheffner M. 2008; Ubiquitin ligase E6-AP and its role in human disease. Biochem Soc Trans 36:797–801 [View Article][PubMed]
     [Google Scholar]
  33. Matsuzawa S. I., Reed J. C. 2001; Siah-1, SIP, and Ebi collaborate in a novel pathway for β-catenin degradation linked to p53 responses. Mol Cell 7:915–926 [View Article][PubMed]
     [Google Scholar]
  34. Mosimann C., Hausmann G., Basler K. 2009; β-Catenin hits chromatin: regulation of Wnt target gene activation. Nat Rev Mol Cell Biol 10:276–286 [View Article][PubMed]
     [Google Scholar]
  35. Nastasi T., Bongiovanni A., Campos Y., Mann L., Toy J. N., Bostrom J., Rottier R., Hahn C., Conaway J. W. et al. 2004; Ozz-E3, a muscle-specific ubiquitin ligase, regulates β-catenin degradation during myogenesis. Dev Cell 6:269–282 [View Article][PubMed]
     [Google Scholar]
  36. Nasu J., Murakami K., Miyagawa S., Yamashita R., Ichimura T., Wakita T., Hotta H., Miyamura T., Suzuki T. et al. 2010; E6AP ubiquitin ligase mediates ubiquitin-dependent degradation of peroxiredoxin 1. J Cell Biochem 111:676–685 [View Article][PubMed]
     [Google Scholar]
  37. Nuber U., Schwarz S. E., Scheffner M. 1998; The ubiquitin-protein ligase E6-associated protein (E6-AP) serves as its own substrate. Eur J Biochem 254:643–649 [View Article][PubMed]
     [Google Scholar]
  38. Oda H., Kumar S., Howley P. M. 1999; Regulation of the Src family tyrosine kinase Blk through E6AP-mediated ubiquitination. Proc Natl Acad Sci U S A 96:9557–9562 [View Article][PubMed]
     [Google Scholar]
  39. Pereira-Suárez A. L., Meraz M. A., Lizano M., Estrada-Chávez C., Hernández F., Olivera P., Pérez E., Padilla P., Yaniv M. et al. 2002; Frequent alterations of the β-catenin protein in cancer of the uterine cervix. Tumour Biol 23:45–53 [View Article][PubMed]
     [Google Scholar]
  40. Ramamoorthy S., Nawaz Z. 2008; E6-associated protein (E6-AP) is a dual function coactivator of steroid hormone receptors. Nucl Recept Signal 6:e006 [View Article][PubMed]
     [Google Scholar]
  41. Reya T., Clevers H. 2005; Wnt signalling in stem cells and cancer. Nature 434:843–850 [View Article][PubMed]
     [Google Scholar]
  42. Rodríguez-Sastre M. A., González-Maya L., Delgado R., Lizano M., Tsubaki G., Mohar A., García-Carrancá A. 2005; Abnormal distribution of E-cadherin and β-catenin in different histologic types of cancer of the uterine cervix. Gynecol Oncol 97:330–336 [View Article][PubMed]
     [Google Scholar]
  43. Scheffner M., Kumar S. 2014; Mammalian HECT ubiquitin-protein ligases: biological and pathophysiological aspects. Biochim Biophys Acta 1843:61–74 [View Article][PubMed]
     [Google Scholar]
  44. 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 [View Article][PubMed]
     [Google Scholar]
  45. Shimoji T., Murakami K., Sugiyama Y., Matsuda M., Inubushi S., Nasu J., Shirakura M., Suzuki T., Wakita T. et al. 2009; Identification of annexin A1 as a novel substrate for E6AP-mediated ubiquitylation. J Cell Biochem 106:1123–1135 [View Article][PubMed]
     [Google Scholar]
  46. Shinohara A., Yokoyama Y., Wan X., Takahashi Y., Mori Y., Takami T., Shimokawa K., Tamaya T. 2001; Cytoplasmic/nuclear expression without mutation of exon 3 of the β-catenin gene is frequent in the development of the neoplasm of the uterine cervix. Gynecol Oncol 82:450–455 [View Article][PubMed]
     [Google Scholar]
  47. Sominsky S., Kuslansky Y., Shapiro B., Jackman A., Haupt Y., Rosin-Arbesfeld R., Sherman L. 2014; HPV16 E6 and E6AP differentially cooperate to stimulate or augment Wnt signaling. Virology 468–470:510–523 [View Article][PubMed]
     [Google Scholar]
  48. Tauriello D. V., Maurice M. M. 2010; The various roles of ubiquitin in Wnt pathway regulation. Cell Cycle 9:3700–3709 [View Article][PubMed]
     [Google Scholar]
  49. Tauriello D. V., Haegebarth A., Kuper I., Edelmann M. J., Henraat M., Canninga-van Dijk M. R., Kessler B. M., Clevers H., Maurice M. M. 2010; Loss of the tumor suppressor CYLD enhances Wnt/β-catenin signaling through K63-linked ubiquitination of Dvl. Mol Cell 37:607–619 [View Article][PubMed]
     [Google Scholar]
  50. Teo J. L., Kahn M. 2010; The Wnt signaling pathway in cellular proliferation and differentiation: a tale of two coactivators. Adv Drug Deliv Rev 62:1149–1155 [View Article][PubMed]
     [Google Scholar]
  51. Tomaić V., Pim D., Banks L. 2009; The stability of the human papillomavirus E6 oncoprotein is E6AP dependent. Virology 393:7–10 [View Article][PubMed]
     [Google Scholar]
  52. Tomai V., Pim D., Thomas M., Massimi P., Myers M. P., Banks L. 2011; Regulation of the human papillomavirus type 18 E6/E6AP ubiquitin ligase complex by the HECT domain-containing protein EDD. J Virol 85:3120–3127 [View Article][PubMed]
     [Google Scholar]
  53. Tran H., Bustos D., Yeh R., Rubinfeld B., Lam C., Shriver S., Zilberleyb I., Lee M. W., Phu L. et al. 2013; HectD1 E3 ligase modifies adenomatous polyposis coli (APC) with polyubiquitin to promote the APC-axin interaction. J Biol Chem 288:3753–3767 [View Article][PubMed]
     [Google Scholar]
  54. Ueda M., Gemmill R. M., West J., Winn R., Sugita M., Tanaka N., Ueki M., Drabkin H. A. 2001; Mutations of the β- and γ-catenin genes are uncommon in human lung, breast, kidney, cervical and ovarian carcinomas. Br J Cancer 85:64–68 [View Article][PubMed]
     [Google Scholar]
  55. Uren A., Fallen S., Yuan H., Usubütün A., Küçükali T., Schlegel R., Toretsky J. A. 2005; Activation of the canonical Wnt pathway during genital keratinocyte transformation: a model for cervical cancer progression. Cancer Res 65:6199–6206 [View Article][PubMed]
     [Google Scholar]
  56. van Amerongen R., Nusse R. 2009; Towards an integrated view of Wnt signaling in development. Development 136:3205–3214 [View Article][PubMed]
     [Google Scholar]
  57. Willert K., Jones K. A. 2006; Wnt signaling: is the party in the nucleus?. Genes Dev 20:1394–1404 [View Article][PubMed]
     [Google Scholar]
  58. Winer I. S., Bommer G. T., Gonik N., Fearon E. R. 2006; Lysine residues Lys-19 and Lys-49 of β-catenin regulate its levels and function in T cell factor transcriptional activation and neoplastic transformation. J Biol Chem 281:26181–26187 [View Article][PubMed]
     [Google Scholar]
  59. Wolyniec K., Levav-Cohen Y., Jiang Y. H., Haupt S., Haupt Y. 2013; The E6AP E3 ubiquitin ligase regulates the cellular response to oxidative stress. Oncogene 32:3510–3519 [View Article][PubMed]
     [Google Scholar]
  60. Zheng L., Ding H., Lu Z., Li Y., Pan Y., Ning T., Ke Y. 2008; E3 ubiquitin ligase E6AP-mediated TSC2 turnover in the presence and absence of HPV16 E6. Genes Cells 13:285–294 [View Article][PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.000624
Loading
Ubiquitin ligase E6AP mediates nonproteolytic polyubiquitylation of β-catenin independent of the E6 oncoprotein
J. Gen. Virol. 97, 3313 (2016); https://doi.org/10.1099/jgv.0.000624
/content/journal/jgv/10.1099/jgv.0.000624
/content/journal/jgv/10.1099/jgv.0.000624
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Most cited Most Cited RSS feed

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