, Volume 11, Issue 12, pp 2147–2157

Uev1A, a ubiquitin conjugating enzyme variant, inhibits stress-induced apoptosis through NF-κB activation

  • Noor A. Syed
  • Parker L. Andersen
  • Robert C. Warrington
  • Wei Xiao


We have previously shown that UEV1 is up-regulated in all tumor cell lines examined and when SV40-transformed human embryonic kidney cells undergo immortalization; however, it is unclear whether and how UEV1 plays a critical role in this process. UEV1A encodes a ubiquitin conjugating enzyme variant, which is required for Ubc13 (ubiquitin conjugating enzyme) catalyzed poly-ubiquitination of target proteins through Lys63-linked chains. One of the target proteins is NEMO/IKKγ (nuclear factor-κB essential modulator/inhibitor of κB protein kinase), a regulatory subunit of IκB kinase in the NF-κB signaling pathway. In this report, we show that constitutive high-level expression of UEV1A alone in cultured human cells was sufficient to cause a significant increase in NF-κB activity as well as the expression of its target anti-apoptotic protein, Bcl-2 (B-cell leukemia/lymphoma 2). Overexpression of UEV1A also conferred prolonged cell survival under serum-deprived conditions, and protected cells against apoptosis induced by diverse stressing agents. All of the effects of Uev1A were reversible upon suppression of UEV1 expression by RNA interference. Our observations presented in this report provide evidence that Uev1A is a critical regulatory component in the NF-κB signaling pathway in response to environmental stresses and identify UEV1A as a potential proto-oncogene.


Uev1A-Ubc13 Lys-63 poly-ubiquitination NF-κB Cancer Apoptosis 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Rothofsky ML, Lin SL (1997) CROC-1 encodes a protein which mediates transcriptional activation of the human FOS promoter. Gene 195:141–49CrossRefPubMedGoogle Scholar
  2. 2.
    Sancho E, Vila MR, Sanchez-Pulido L et al (1998) Role of UEV-1, an inactive variant of the E2 ubiquitin-conjugating enzymes, in in vitro differentiation and cell cycle behavior of HT-29-M6 intestinal mucosecretory cells. Mol Cell Biol 18:576–89PubMedGoogle Scholar
  3. 3.
    Ma L, Broomfield S, Lavery C et al (1998) Up-regulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines. Oncogene 17:1321–326CrossRefPubMedGoogle Scholar
  4. 4.
    Broomfield S, Chow BL, Xiao W (1998) MMS2, encoding a ubiquitin-conjugating-enzyme-like protein, is a member of the yeast error-free postreplication repair pathway. Proc Natl Acad Sci USA 95:5678–683CrossRefPubMedGoogle Scholar
  5. 5.
    Xiao W, Lin SL, Broomfield S, Chow BL, Wei YF (1998) The products of the yeast MMS2 and two human homologs (hMMS2 and CROC-1) define a structurally and functionally conserved Ubc-like protein family. Nucleic Acids Res 26:3908–914CrossRefPubMedGoogle Scholar
  6. 6.
    Kallioniemi A, Kallioniemi OP, Piper J et al (1994) Detection and mapping of amplified DNA sequences in breast cancer by comparative genomic hybridization. Proc Natl Acad Sci USA 91:2156–160CrossRefPubMedGoogle Scholar
  7. 7.
    Tanner MM, Tirkkonen M, Kallioniemi A et al (1994) Increased copy number at 20q13 in breast cancer: defining the critical region and exclusion of candidate genes. Cancer Res 54:4257–260PubMedGoogle Scholar
  8. 8.
    Tanner MM, Tirkkonen M, Kallioniemi A et al (1995) Amplification of chromosomal region 20q13 in invasive breast cancer: prognostic implications. Clin Cancer Res 1:1455–461PubMedGoogle Scholar
  9. 9.
    Brinkmann U, Gallo M, Polymeropoulos MH, Pastan I (1996) The human CAS (cellular apoptosis susceptibility) gene mapping on chromosome 20q13 is amplified in BT474 breast cancer cells and part of aberrant chromosomes in breast and colon cancer cell lines. Genome Res 6:187–94CrossRefPubMedGoogle Scholar
  10. 10.
    El-Rifai W, Harper JC, Cummings OW et al (1998) Consistent genetic alterations in xenografts of proximal stomach and gastro-esophageal junction adenocarcinomas. Cancer Res 58:34–7PubMedGoogle Scholar
  11. 11.
    Savelieva E, Belair CD, Newton MA et al (1997) 20q gain associates with immortalization: 20q13.2 amplification correlates with genome instability in human papillomavirus 16 E7 transformed human uroepithelial cells. Oncogene 14:551–60CrossRefPubMedGoogle Scholar
  12. 12.
    Yeager TR, DeVries S, Jarrard DF et al (1998) Overcoming cellular senescence in human cancer pathogenesis. Genes Dev 12:163–74CrossRefPubMedGoogle Scholar
  13. 13.
    McKenna S, Spyracopoulos L, Moraes T et al (2001) Noncovalent interaction between ubiquitin and the human DNA repair protein Mms2 is required for Ubc13-mediated polyubiquitination. J Biol Chem 276:40120–0126CrossRefPubMedGoogle Scholar
  14. 14.
    McKenna S, Moraes T, Pastushok L et al (2003) An NMR-based model of the ubiquitin-bound human ubiquitin conjugation complex Mms2.Ubc13. The structural basis for lysine 63 chain catalysis. J Biol Chem 278:13151–3158CrossRefPubMedGoogle Scholar
  15. 15.
    Moraes TF, Edwards RA, McKenna S et al (2001) Crystal structure of the human ubiquitin conjugating enzyme complex, hMms2-hUbc13. Nat Struct Biol 8:669–73CrossRefPubMedGoogle Scholar
  16. 16.
    Hofmann RM, Pickart CM (1999) Noncanonical MMS2-encoded ubiquitin-conjugating enzyme functions in assembly of novel polyubiquitin chains for DNA repair. Cell 96:645–53CrossRefPubMedGoogle Scholar
  17. 17.
    Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–33CrossRefPubMedGoogle Scholar
  18. 18.
    Deng L, Wang C, Spencer E et al (2000) Activation of the IκB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103:351–61CrossRefPubMedGoogle Scholar
  19. 19.
    Wang C, Deng L, Hong M et al (2001) TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412:346–51CrossRefPubMedGoogle Scholar
  20. 20.
    Shi CS, Kehrl JH (2003) Tumor necrosis factor (TNF)-induced germinal center kinase-related (GCKR) and stress-activated protein kinase (SAPK) activation depends upon the E2/E3 complex Ubc13-Uev1A/TNF receptor-associated factor 2 (TRAF2). J Biol Chem 278:15429–5434CrossRefPubMedGoogle Scholar
  21. 21.
    Lin A, Karin M (2003) NF-κB in cancer: a marked target. Semin Cancer Biol 13:107–14CrossRefPubMedGoogle Scholar
  22. 22.
    Hayden MS, Ghosh S (2004) Signaling to NF-κB. Genes Dev 18:2195–224CrossRefPubMedGoogle Scholar
  23. 23.
    Zhou H, Wertz I, O’Rourke K et al (2004) Bcl10 activates the NF-κB pathway through ubiquitination of NEMO. Nature 427:167–71CrossRefPubMedGoogle Scholar
  24. 24.
    Sun L, Deng L, Ea CK, Xia ZP, Chen ZJ (2004) The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol Cell 14:289–01CrossRefPubMedGoogle Scholar
  25. 25.
    Ea CK, Deng L, Xia ZP, Pineda G, Chen ZJ (2006) Activation of IKK by TNFα requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol Cell 22:245–57CrossRefPubMedGoogle Scholar
  26. 26.
    Debatin KM (2004) Apoptosis pathways in cancer and cancer therapy. Cancer Immunol Immunother 53:153–59CrossRefPubMedGoogle Scholar
  27. 27.
    Azoitei N, Wirth T, Baumann B (2005) Activation of the IκB kinase complex is sufficient for neuronal differentiation of PC12 cells. J Neurochem 93:1487–501CrossRefPubMedGoogle Scholar
  28. 28.
    Chen GG, Liang NC, Lee JF et al (2004) Over-expression of Bcl-2 against Pteris semipinnata L-induced apoptosis of human colon cancer cells via a NF-κB-related pathway. Apoptosis 9:619–27CrossRefPubMedGoogle Scholar
  29. 29.
    Chen GG, Lee JF, Wang SH et al (2002) Apoptosis induced by activation of peroxisome-proliferator activated receptor-gamma is associated with Bcl-2 and NF-κB in human colon cancer. Life Sci 70:2631–646CrossRefPubMedGoogle Scholar
  30. 30.
    O’Connell J, Bennett MW, Nally K et al (2000) Altered mechanisms of apoptosis in colon cancer: Fas resistance and counterattack in the tumor-immune conflict. Ann N Y Acad Sci 910:178–92PubMedCrossRefGoogle Scholar
  31. 31.
    Ditsworth D, Zong WX (2004) NF-κB: Key mediator of inflammation-associated cancer. Cancer Biol Ther 3:1214–216PubMedCrossRefGoogle Scholar
  32. 32.
    Pikarsky E, Porat RM, Stein I et al (2004) NF-κB functions as a tumour promoter in inflammation-associated cancer. Nature 431:461–66CrossRefPubMedGoogle Scholar
  33. 33.
    Clevers H (2004) At the crossroads of inflammation and cancer. Cell 118:671–74CrossRefPubMedGoogle Scholar
  34. 34.
    Karin M (2005) Inflammation and cancer: the long reach of Ras. Nat Med 11:20–1CrossRefPubMedGoogle Scholar
  35. 35.
    Yu JY, DeRuiter SL, Turner DL (2002) RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 99:6047–052CrossRefPubMedGoogle Scholar
  36. 36.
    Andersen PL, Zhou H, Pastushok L et al (2005) Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A. J Cell Biol 170:745–55CrossRefPubMedGoogle Scholar
  37. 37.
    Warrington RC, Norum JN, Hilchey JL, Watt C, Fang WD (2003) A simple, informative, and quantitative flow cytometric method for assessing apoptosis in cultured cells. Prog Neuropsychopharmacol Biol Psychiatry 27:231–43CrossRefPubMedGoogle Scholar
  38. 38.
    Ke N, Albers A, Claassen G et al (2004) One-week 96-well soft agar growth assay for cancer target validation. Biotechniques 36:826–28, 830PubMedGoogle Scholar
  39. 39.
    Li Z, Xiao W, McCormick JJ, Maher VM (2002) Identification of a protein essential for a major pathway used by human cells to avoid UV- induced DNA damage. Proc Natl Acad Sci USA 99:4459–464CrossRefPubMedGoogle Scholar
  40. 40.
    Tamatani M, Che YH, Matsuzaki H et al (1999) Tumor necrosis factor induces Bcl-2 and Bcl-x expression through NF-κB activation in primary hippocampal neurons. J Biol Chem 274:8531–538CrossRefPubMedGoogle Scholar
  41. 41.
    Tamatani M, Mitsuda N, Matsuzaki H et al (2000) A pathway of neuronal apoptosis induced by hypoxia/reoxygenation: roles of nuclear factor-κB and Bcl-2. J Neurochem 75:683–93CrossRefPubMedGoogle Scholar
  42. 42.
    Takehara T, Liu X, Fujimoto J, Friedman SL, Takahashi H (2001) Expression and role of Bcl-xL in human hepatocellular carcinomas. Hepatology 34:55–1CrossRefPubMedGoogle Scholar
  43. 43.
    Emanuele S, Calvaruso G, Lauricella M et al (2002) Apoptosis induced in hepatoblastoma HepG2 cells by the proteasome inhibitor MG132 is associated with hydrogen peroxide production, expression of Bcl-XS and activation of caspase-3. Int J Oncol 21:857–65PubMedGoogle Scholar
  44. 44.
    Giuliano M, Bellavia G, Lauricella M et al (2004) Staurosporine-induced apoptosis in Chang liver cells is associated with down-regulation of Bcl-2 and Bcl-XL. Int J Mol Med 13:565–71PubMedGoogle Scholar
  45. 45.
    Chiao PJ, Na R, Niu J et al (2002) Role of Rel/NF-κB transcription factors in apoptosis of human hepatocellular carcinoma cells. Cancer 95:1696–705CrossRefPubMedGoogle Scholar
  46. 46.
    Champoux JJ (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70:369–13CrossRefPubMedGoogle Scholar
  47. 47.
    Pommier Y, Pourquier P, Fan Y, Strumberg D (1998) Mechanism of action of eukaryotic DNA topoisomerase I and drugs targeted to the enzyme. Biochim Biophys Acta 1400:83–05PubMedGoogle Scholar
  48. 48.
    Weil M, Jacobson MD, Coles HS et al (1996) Constitutive expression of the machinery for programmed cell death. J Cell Biol 133:1053–059CrossRefPubMedGoogle Scholar
  49. 49.
    Miyamoto Y, Takikawa Y, De Lin S, Sato S, Suzuki K (2004) Apoptotic hepatocellular carcinoma HepG2 cells accelerate blood coagulation. Hepatol Res 29:167–72CrossRefPubMedGoogle Scholar
  50. 50.
    Raff MC (1992) Social controls on cell survival and cell death. Nature 356:397–00CrossRefPubMedGoogle Scholar
  51. 51.
    Izuishi K, Kato K, Ogura T, Kinoshita T, Esumi H (2000) Remarkable tolerance of tumor cells to nutrient deprivation: possible new biochemical target for cancer therapy. Cancer Res 60:6201–207PubMedGoogle Scholar
  52. 52.
    Karin M, Ben-Neriah Y (2000) Phosphorylation meets ubiquitination: the control of NF-κB activity. Annu Rev Immunol 18:621–63CrossRefPubMedGoogle Scholar
  53. 53.
    Huang TT, Wuerzberger-Davis SM, Wu ZH, Miyamoto S (2003) Sequential modification of NEMO/IKKγ by SUMO-1 and ubiquitin mediates NF-κB activation by genotoxic stress. Cell 115:565–76CrossRefPubMedGoogle Scholar
  54. 54.
    Kracklauer MP, Schmidt C (2003) At the crossroads of SUMO and NF-κB. Mol Cancer 2:39CrossRefPubMedGoogle Scholar
  55. 55.
    Greten FR, Eckmann L, Greten TF et al (2004) IKKβ links inflammation and tumorigenesis in a mouse model of colitis-associated cancer. Cell 118:285–96CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science + Business Media, LLC 2006

Authors and Affiliations

  • Noor A. Syed
    • 1
  • Parker L. Andersen
    • 1
  • Robert C. Warrington
    • 2
  • Wei Xiao
    • 1
  1. 1.Department of Microbiology and ImmunologyUniversity of SaskatchewanSaskatoonCanada
  2. 2.Department of BiochemistryUniversity of SaskatchewanSaskatoonCanada

Personalised recommendations