Downregulation of Bax mRNA expression and protein stability by the E6 protein of human papillomavirus 16 Free

Abstract

Previous studies have shown that human papillomavirus (HPV) 16 E6 inhibits apoptosis induced during terminal differentiation of primary human keratinocytes (PHKs) triggered by serum and calcium. E6 inhibition of apoptosis was accompanied with prolonged expression of Bcl-2 and reduced elevation of Bax levels. In the present study, the effect of E6 on Bax mRNA expression and protein stability was investigated. These studies indicate that stable E6 expression in differentiating keratinocytes reduced the steady-state levels of Bax mRNA and shortened the half-life of Bax protein. These results were confirmed in transiently transfected 293T cells where E6 degraded Bax in a dose-dependent manner. Bax degradation was also exhibited in Saos-2 cells that lack p53, indicating its p53 independence. E6 did not form complexes with Bax and did not induce Bax degradation under experimental conditions where p53 was degraded. Finally, E6 aa 120–132 were shown to be necessary for Bax destabilization and, more importantly, for abrogating the ability of Bax to induce cellular apoptosis, highlighting the functional consequences of the E6-induced alterations in Bax expression.

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2005-03-01
2024-03-29
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References

  1. Alfandari J., Shnitman Magal S., Jackman A., Schlegel R., Gonen P., Sherman L. 1999; HPV16 E6 oncoprotein inhibits apoptosis induced during serum-calcium differentiation of foreskin human keratinocytes. Virology 257:383–396 [CrossRef]
    [Google Scholar]
  2. Bargou R. C., Daniel P. T., Mapara M. Y., Bommert K., Wagener C., Kallinich B., Royer H. D., Dorken B. 1995; Expression of the bcl-2 gene family in normal and malignant breast tissue: low bax-alpha expression in tumor cells correlates with resistance towards apoptosis. Int J Cancer 60:854–859 [CrossRef]
    [Google Scholar]
  3. Budtz P. E. 1994; Epidermal homeostasis: a new model that includes apoptosis. In Apoptosis II: the Molecular Basis of Apoptosis in Disease pp  165–184 Edited by Tomei L. D., Cope F. O. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  4. Burd E. M. 2003; Human papillomavairus and cervical cancer. Clin Microbiol Rev 16:1–17 [CrossRef]
    [Google Scholar]
  5. Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C. 1994; Green fluorescent protein as a marker for gene expression. Science 263:802–805 [CrossRef]
    [Google Scholar]
  6. Chang Y.-C., Lee Y.-S., Tejima T., Tanaka K., Omura S., Heintz N. H., Mitsui Y., Magae J. 1998; mdm2 and bax, downstream mediators of the p53 response, are degraded by the ubiquitin-proteasome pathway. Cell Growth Differ 9:79–84
    [Google Scholar]
  7. Chen J. J., Reid C. E., Band V., Androphy E. J. 1995; Interaction of papillomavirus E6 oncoproteins with a putative calcium-binding protein. Science 269:529–531 [CrossRef]
    [Google Scholar]
  8. Degenhardt Y.-Y., Silverstein S. J. 2001; Gps2, a protein partner for human papillomavirus E6 protein. J Virol 75:151–160 [CrossRef]
    [Google Scholar]
  9. Degli Esposti M., Dive C. 2003; Mitochondrial membrane permeabization by Bax/Bak. Biochem Biophys Res Commun 304:455–461 [CrossRef]
    [Google Scholar]
  10. Delehedde M., Cho S. H., Sarkiss M., Brisbay S., Davies M., El-Naggar A. K., McDonnell T. J. 1999; Altered expression of bcl-2 family member proteins in nonmelanoma skin cancer. Cancer 85:1514–1522 [CrossRef]
    [Google Scholar]
  11. Dewson G., Snowden R. T., Almond J. B., Dyer M. J. S., Cohen G. M. 2003; Conformational change and mitochondrial translocation of Bax accompany proteasome inhibitor-induced apoptosis of chronic lymphocytic leukemic cells. Oncogene 22:2643–2654 [CrossRef]
    [Google Scholar]
  12. Eckert R. L., Green H. 1986; Structure and evolution of the human involucrin gene. Cell 46:583–589 [CrossRef]
    [Google Scholar]
  13. Filippova M., Song H., Connolly J. L., Dermody T. S., Duerksen-Hughes P. J. 2002; The human papillomavirus 16 E6 protein binds to tumor necrosis factor (TNF) R1 and protects cells from TNF-induced apoptosis. J Biol Chem 277:21730–21739 [CrossRef]
    [Google Scholar]
  14. Gandarillas A., Goldsmith L. A., Gschmeissner S., Leigh I. M., Watt F. M. 1999; Evidence that apoptosis and terminal differentiation of epidermal keratinocytes are distinct processes. Exp Dermatol 8:71–79 [CrossRef]
    [Google Scholar]
  15. Gao G., Dou Q. P. 2000; N-terminal cleavage of Bax by calpain generates a potent proapoptotic 18-kDa fragment that promotes Bcl-2-independent cytochrome c release and apoptotic cell death. J Cell Biochem 80:53–72
    [Google Scholar]
  16. Gao Q., Srinivasan S., Boyer S. N., Wazer D. E., Band V. 1999; The E6 oncoprotein of high-risk papillomaviruses bind to a novel putative GAP protein E6TP1, and target it for degradation. Mol Cell Biol 19:733–744
    [Google Scholar]
  17. Gao Q., Kumar A., Srinivasan S., Singh L., Mukai H., Ono Y., Wazer D. E., Band V. 2000; PKN binds and phosphorylates human papillomavirus E6 oncoprotein. J Biol Chem 275:14824–14830 [CrossRef]
    [Google Scholar]
  18. Gewin L., Galloway D. 2001; E-Box-dependent activation of telomerase by human papillomavirus, type 16 E6 does not require induction of c- myc . J Virol 75:7198–7201 [CrossRef]
    [Google Scholar]
  19. Glaunsinger B. A., Lee S. S., Thomas M., Banks L., Javier R. 2000; Interaction of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and high-risk papillomavirus E6 oncoproteins. Oncogene 19:5270–5280 [CrossRef]
    [Google Scholar]
  20. Gross-Mesilaty S., Reinstein E., Bercovich B., Tobias K. E., Schwartz A. L., Kahana C., Ciechanover A. 1998; Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway. Proc Natl Acad Sci U S A 95:8058–8063 [CrossRef]
    [Google Scholar]
  21. Han J., Modha D., White E. 1998; Interaction of E1B 19K with Bax is required to block Bax-induced loss of mitochrondrial membrane potential and apoptosis. Oncogene 17:2993–3005 [CrossRef]
    [Google Scholar]
  22. Hawley-Nelson P., Vousden K. H., Hubbert N. L., Lowy D. R., Schiller J. T. 1989; HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J 8:3905–3910
    [Google Scholar]
  23. Hockenbery D. M., Zutter M., Hickey W., Nahm M., Korsmeyer S. J. 1991; BCL2 protein is topographically restricted in tissues characterized by apoptotic cell death. Proc Natl Acad Sci U S A 88:6961–6965 [CrossRef]
    [Google Scholar]
  24. Huibregtse J. M., Scheffner M., Howley P. M. 1991; A cellular protein mediates association of p53 with E6 oncoprotein of human papillomavirus tyes 16 or 18. EMBO J 10:4129–4135
    [Google Scholar]
  25. Huibregtse J. M., Scheffner M., Howley P. M. 1993; Cloning and expression of the cDNA for E6-AP, a protein that mediates the interaction of the human papilllomavirus E6 oncoprotein with p53. Mol Cell Biol 13:775–784
    [Google Scholar]
  26. Jordan R. C. K., Catzavelos G. C., Barrett A. W., Speight P. M. 1996; Differential expression of bcl-2 and bax in squamous cell carcinomas of the oral cavity. Eur J Cancer B Oral Oncol 32B:394–400
    [Google Scholar]
  27. Kitanaka C., Namiki T., Noguchi K. 7 other authors 1997; Caspase-dependent apoptosis of COS-7 cells induced by Bax overexpression: differential effects of Bcl-2 and Bcl-xL on Bax-induced caspase activation and apoptosis. Oncogene 15:1763–1772 [CrossRef]
    [Google Scholar]
  28. Kiyono T., Hiraiwa H., Fujita M., Hayashi Y., Akiyama T., Ishibashi M. 1997; Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein. Proc Natl Acad Sci U S A 94:11612–11616 [CrossRef]
    [Google Scholar]
  29. Kuhne C., Banks L. 1998; E3-ubiquitin ligase/E6-AP links multicopy maintenance protein 7 to the ubiquitination pathway by a novel motif, the L2G Box. J Biol Chem 273:34302–34309 [CrossRef]
    [Google Scholar]
  30. Kukimoto I., Aihara S., Yoshiike K., Kanda T. 1998; Human papillomavirus oncoprotein E6 binds to the C-terminal region of human minichromosome maintenance 7 protein. Biochem Biophys Res Commun 249:258–262 [CrossRef]
    [Google Scholar]
  31. Kumar A., Zhao Y., Meng G. 9 other authors 2002; Human papillomavirus oncoprotein E6 inactivates the transcriptional coactivator human ADA3. Mol Cell Biol 22:5801–5812 [CrossRef]
    [Google Scholar]
  32. Lee S. S., Weiss R. S., Javier R. T. 1997; Binding of human virus oncoproteins to hDlg/SAP97, a mammalian homolog of the Drosophila discs large tumor suppressor protein. Proc Natl Acad Sci U S A 94:6670–6675 [CrossRef]
    [Google Scholar]
  33. Lee S. S., Glaunsinger B., Mantovani F., Banks L., Javier R. T. 2000; Multi-PDZ domain protein MUPP1 is a cellular target for both adenovirus E4-ORF1 and high-risk papillomavirus type 18 E6 oncoproteins. J Virol 74:9680–9693 [CrossRef]
    [Google Scholar]
  34. Li B., Dou Q. P. 2000; Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. Proc Natl Acad Sci U S A 97:3850–3855 [CrossRef]
    [Google Scholar]
  35. Mantovani F., Banks L. 2001; The human papillomavirus E6 protein and its contribution to malignant progression. Oncogene 20:7874–7887 [CrossRef]
    [Google Scholar]
  36. Marshall W. L., Yim C., Gustafson E., Graf T., Sage D. R., Hanify K., Williams L., Fingeroth J., Finberg R. W. 1999; Epstein-Barr virus encodes a novel homolog of the bcl-2 oncogene that inhibits apoptosis and associates with Bax and Bak. J Virol 73:5181–5185
    [Google Scholar]
  37. Marshansky V., Wang X., Bertrand R., Luo H., Duguid W., Chinnadurai G., Kanaan N., Vu M. D., Wu J. 2001; Proteasomes modulate balance among proapoptotic and antiapoptotic Bcl-2 family members and compromise functioning of the electron transport chain in leukemic cells. J Immunol 166:3130–3142 [CrossRef]
    [Google Scholar]
  38. Maruoka Y., Harada H., Mitsuyasu T., Seta Y., Kurokawa H., Kajiyama M., Toyoshima K. 1997; Keratinocytes become terminally differentiated in a process involving programmed cell death. Biochem Biophys Res Commun 238:886–890 [CrossRef]
    [Google Scholar]
  39. Mitchell K. O., Ricci M. S., Miyashita T., Dicker D. T., Jin Z., Reed J. C., El-Deiry W. S. 2000; Bax is a transcriptional target and mediator of c-Myc-induced apoptosis. Cancer Res 60:6318–6325
    [Google Scholar]
  40. Mitra R. S., Wrone-Smith T., Simonian P., Foreman K. E., Nunez G., Nickoloff B. J. 1997; Apoptosis in keratinocytes is not dependent on induction of differentiation. Lab Invest 76:99–107
    [Google Scholar]
  41. Miyashita T., Reed J. C. 1995; Tumor suppressor p53 is a direct transcriptional activator of the human bax gene. Cell 80:293–299 [CrossRef]
    [Google Scholar]
  42. Miyashita T., Krajewski S., Krajewska M., Wang H. G., Lin H. K., Leibermann D. A., Hoffman B., Reed J. C. 1994; Tumor suppressor p53 is a regulator of bcl-2 and bax gene expression in vitro and in vivo . Oncogene 9:1799–1805
    [Google Scholar]
  43. Munger K., Howley P. M. 2002; Human papillomavirus immortalizaton and transformation functions. Virus Res 89:213–228 [CrossRef]
    [Google Scholar]
  44. Nakagawa S., Huibregtse J. M. 2000; Human scribble (Vartul) is targeted for ubiquitin-mediated degradation by the high-risk papillomavirus E6 proteins and the E6AP ubiquitin-protein ligase. Mol Cell Biol 20:8244–8253 [CrossRef]
    [Google Scholar]
  45. Oh S. T., Kyo S., Laimins L. A. 2001; Telomerase activation by human papillomavirus type 16 E6 protein: induction of human telomerase reverse transcriptase expression through Myc and GC-rich Sp1 binding sites. J Virol 75:5559–5566 [CrossRef]
    [Google Scholar]
  46. Patel D., Huang S. M., Baglia L. A., McCance D. J. 1999; The E6 protein of human papillomavirus type 16 binds to and inhibits co-activation by CBP and p300. EMBO J 18:5061–5072 [CrossRef]
    [Google Scholar]
  47. Perez D., White E. 2000; TNF- α signals apoptosis through a bid-dependent conformational change in bax that is inhibited by E1B 19K. Mol Cell 6:53–63 [CrossRef]
    [Google Scholar]
  48. Polakowska R. R., Piacentini M., Barlett R., Goldsmith L. A., Haake A. R. 1994; Apoptosis in human skin development: morphogenesis periderm and stem cells. Dev Dyn 199:176–188 [CrossRef]
    [Google Scholar]
  49. Reed J. C. 1998; Bcl-2 family proteins. Oncogene 17:3225–3236
    [Google Scholar]
  50. Ronco L. V., Karpova A. Y., Vidal M., Howley P. M. 1998; Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity. Genes Dev 12:2061–2072 [CrossRef]
    [Google Scholar]
  51. Scheffner M., Whitaker N. J. 2003; Human papillomavirus-induced carcinogenesis and the ubiquitin-proteasome system. Semin Cancer Biol 13:59–67 [CrossRef]
    [Google Scholar]
  52. Scheffner M., Werness B. A., Huibregtse J. M., Levine A. J., Howley P. M. 1990; The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell 63:1129–1136 [CrossRef]
    [Google Scholar]
  53. Schlegel R., Phelps W. C., Zhang Y.-L., Barbosa M. 1988; Quantitative keratinocyte assay detects two biological activities of human papillomavirus and identifies viral types associated with cervical carcinoma. EMBO J 7:3181–3187
    [Google Scholar]
  54. Scorrano L., Korsmeyer S. J. 2003; Mechanisms of cytochrome c release by proapoptotic BCL-2 family members. Biochem Biophys Res Commun 304:437–444 [CrossRef]
    [Google Scholar]
  55. Sherman L., Schlegel R. 1996; Serum- and calcium-induced differentiation of human keratinocytes is inhibited by the E6 oncoprotein of human papillomavirus type 16. J Virol 70:3269–3279
    [Google Scholar]
  56. Sherman L., Gazit A., Yaniv A., Kawakami T., Dahlberg J. E., Tronick S. R. 1988; Localization of sequences responsible for trans-activation of the equine infectious anemia virus long terminal repeat. J Virol 62:120–126
    [Google Scholar]
  57. Sherman L., Jackman A., Itzhaki H., Stoppler M. C., Koval D., Schlegel R. 1997; Inhibition of serum- and calcium-induced differentiation of human keratinocytes by HPV 16 E6 oncoprotein: role of p53 inactivation. Virology 237:296–306 [CrossRef]
    [Google Scholar]
  58. Sherman L., Itzhaki H., Jackman A., Chen J. J., Koval D., Schlegel R. 2002; Inhibition of serum- and calcium-induced terminal differentiation of human keratinocytes by HPV 16 E6: study of the association with p53 degradation, inhibition of p53 transactivation and binding to E6BP. Virology 292:309–320 [CrossRef]
    [Google Scholar]
  59. Srivenugopal K. S., Ali-Osman F. 2002; The DNA repair protein, 06-methylguanine-DNA methyltransferase is a proteolytic target for the E6 human papillomavirus oncoprotein. Oncogene 21:5940–5945 [CrossRef]
    [Google Scholar]
  60. Thomas M., Banks L. 1998; Inhibition of Bak-induced apoptosis by HPV-18 E6. Oncogene 17:2943–2954 [CrossRef]
    [Google Scholar]
  61. Thomas M., Banks L. 1999; Human papillomavirus (HPV) E6 interactions with Bak are conserved amongst E6 proteins from high and low risk HPV types. J Gen Virol 80:1513–1517
    [Google Scholar]
  62. Thomas M., Pim D., Banks L. 1999; The role of the E6-p53 interaction in the molecular pathogenesis of HPV. Oncogene 18:7690–7700 [CrossRef]
    [Google Scholar]
  63. Thomas M., Laura R., Hepner K., Guccione E., Sawyers C., Lasky L., Banks L. 2002; Oncogenic human papillomavirus E6 proteins target the MAGI-2 and MAGI-3 proteins for degradation. Oncogene 21:5088–5096 [CrossRef]
    [Google Scholar]
  64. Thomson B. J. 2001; Viruses and apoptosis. Int J Exp Pathol 82:65–76 [CrossRef]
    [Google Scholar]
  65. Tong X., Howley P. M. 1997; The bovine papillomavirus E6 oncoprotein interacts with paxillin and disrupts the actin cytoskeleton. Proc Natl Acad Sci U S A 94:4412–4417 [CrossRef]
    [Google Scholar]
  66. Tsujimoto Y., Shimizu S. 2000; Bcl-2 family: life-or-death switch. FEBS Lett 466:6–10 [CrossRef]
    [Google Scholar]
  67. Veldman T., Horikawa I., Barrett J. C., Schlegel R. 2001; Transcriptional activation of the telomerase hTERT gene by human papillomavirus type 16 E6 oncoprotein. J Virol 75:4467–4472 [CrossRef]
    [Google Scholar]
  68. Veldman T., Liu X., Yuan H., Schlegel R. 2003; Human papillomavirus E6 and Myc proteins associate in vivo and bind to and cooperatively activate the telomerase reverse-transcriptase. Proc Natl Acad Sci U S A 100:8211–8216 [CrossRef]
    [Google Scholar]
  69. Wood D. E., Thomas A., Devi L. A., Berman Y., Beavis R. C., Reed J. C., Newcomb E. W. 1998; Bax cleavage is mediated by calpain during drug-induced apoptosis. Oncogene 17:1069–1078 [CrossRef]
    [Google Scholar]
  70. Zimmermann H., Degenkolbe R., Bernard H. U., O'Connor M. J. 1999; The human papillomavirus type 16 E6 oncoprotein can down-regulate p53 activity by targeting the transcriptional coactivator CBP/p300. J Virol 73:6209–6219
    [Google Scholar]
  71. Zur-Hausen H. 1996; Papilloma virus infection - a major cause of human cancers. Biochim Biophys Acta 1288F55–78
    [Google Scholar]
  72. Zur-Hausen H. 2002; Papillomaviruses and cancer: from basic studies to clinical applications. Nature 2:342–350
    [Google Scholar]
  73. Zwerschke W., Jansen-Durr P. 2000; Cell transformation by the E7 oncoprotein of human papillomavirus type 16: interactions with nuclear and cytoplasmic target proteins. Adv Cancer Res 78:1–29
    [Google Scholar]
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